Transgenic mice comprising transcription control elements associated with mouse eosinophil peroxidase expression

ABSTRACT

The present invention relates to novel transcription control elements derived from a mouse eosinophil peroxidase gene. The novel transcription control elements described in the disclosure may comprise isolated polynucleotides, expression cassettes, vectors, recombinant cells, and transgenic animals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to U.S. Provisional Patent Application Ser.No. 60/285,603, filed Apr. 20, 2001, from which priority is claimedunder 35 USC §119(e)(1), and which application is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to the field of molecularbiology and medicine. In particular, the invention relates totranscription control elements derived from the genomic locus of themurine eosinophil peroxidase (EPX) gene, and methods of use thereof. Theinvention further relates to isolated polynucleotides in regulatoryregions of the murine EPX gene, to reporter constructs comprising thoseisolated polynucleotides, to cells transformed with those reporterconstructs, and to transgenic animals comprising those reporterconstructs. The invention further relates to in vivo assay methods thatemploy animals transfected with such reporter constructs, and/ortransgenic animals comprising such constructs.

BACKGROUND OF THE INVENTION

Eosinophils play a protective role in host immunity to parasitic worminfections and, detrimentally, are involved in the pathophysiology ofasthma and other allergic diseases. Eosinophils are prominent in airwayinflammation. Eosinophils are involved in diseases like asthma, chroniceosinophilic pneumonia, Churg-Strauss Syndrome, Hypereosinophilicsyndrome, allergic rhinitis, atopic dermatitis.

The present invention relates to constructs and methods to mark theeosinophils in vivo and also to methods for directly monitoring EPX generegulation in real-time in live animals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict nucleotide sequences of transcriptional controlelement from mouse eosinophil peroxidase. FIG. 1A (SEQ ID NO:1)comprises the nucleotide sequence of a transcriptional control elementfrom the mouse eosinophil peroxidase (EPX) gene locus. FIG. 1B (SEQ IDNO:2) comprises an approximately 9.5kb sequence of FIG. 1A (SEQ IDNO:1). In the FIG. 1A, the sequence represents 9,828 nucleotides intotal, the translational start codon (ATG) is located at positions9,826-9,828, a TATA box is located at positions 9,679-9,682, a majortranscription start site begins with the A at position 9,709. A novelapproximately 9.5 kb region of the EPX gene locus is from nucleotideposition 1 to 9,450 of FIG. 1A and the approximately 9.5 kb sequence ispresented alone in FIG. 1B (SEQ ID NO:2).

FIG. 2 presents a restriction map of an XbaI fragment (10,985 basepairs) derived from a BAC-mouse genomic clone that comprises the EPXtranscriptional control elements described herein.

SUMMARY OF THE INVENTION

The present invention relates to novel transcription control elementsderived from the genomic locus of the murine eosinophil peroxidase (EPX)gene. The present invention comprises isolated polynucleotides,expression cassettes, vectors, recombinant cells, and transgenic,non-human animals that comprise the transcription control elementsdescribed herein.

In one aspect, the present invention relates to a transgenic rodentcomprising, an expression cassette wherein the expression cassettecomprises a polynucleotide derived from the mouse eosinophil peroxidasegene, wherein the polynucleotide is operably linked to a coding sequenceof interest. Typically the polynucleotide comprises at least onetranscriptional control element. Further, eosinophils from thetransgenic animal express the coding sequence of interest at a greaterlevel than other, non-eosinophil blood cell-types. The polynucleotidehas, for example, at least 95% or greater identity to nucleotides1-9,825 of SEQ ID NO:1. Alternately, the polynucleotide has at least 95%or greater identity to SEQ ID NO:2, or at least 95% or greater identityto SEQ ID NO:7. In one embodiment, the polynucleotide consists of apolynucleotide having at least 95% or greater identity to nucleotides1-9,825 of SEQ ID NO:1. In a further embodiment, the polynucleotideconsists of a polynucleotide having at least 95% or greater identity toSEQ ID NO:2. In yet another embodiment, the polynucleotide consists of apolynucleotide having at least 95% or greater identity to SEQ ID NO:7.

In another aspect of the present invention, the transgenic rodent showsinduction of the expression of the coding sequence of interest followingovalbumin challenge via intraperitoneal injection or via airwayinhalation.

In another aspect of the present invention, administration of IL-5 tothe transgenic rodent promotes greater trafficking of eosinophils to theesophagus of the transgenic rodent than to other regions of the body ofthe transgenic rodent, and the trafficking is monitored by trackingexpression of the coding sequence of interest.

In a further aspect of the present invention, greater basal expressionof the coding sequence of interest is seen in the lamina propria of thetransgenic rodent relative to basal expression of the coding sequence ofinterest in other regions of the body of the transgenic rodent.

In yet a further aspect of the present invention, expression of thecoding sequence of interest is induced in the transgenic rodent whenIL-5 is over-expressed in the transgenic animal.

Further, in another aspect of the present invention, levels ofexpression of the coding sequence of interest after allergen-inductionare higher before treatment with a glucocorticoid than after treatmentwith the glucocorticoid. One exemplary glucocorticoid is dexamethasone.

The coding sequence of interest in the transgenic rodent may, forexample, be a reporter gene (or reporter sequence). One exemplaryreporter sequence encodes a light-generating protein (e.g., abioluminescent protein or a fluorescent protein). In one embodiment, thereporter sequence encodes the bioluminescent protein luciferase. Inanother embodiment, the reporter sequence encodes a fluorescent protein,including, but not limited to, blue fluorescent protein, cyanfluorescent protein, green fluorescent protein, yellow fluorescentprotein, and/or red fluorescent protein.

The transgenic rodent of the present invention may, for example, be amouse, rat, gerbil, hamster, or guinea pig.

The present invention also includes methods employing the transgenicanimals of the present invention. One exemplary method is foridentifying an analyte that modulates expression of a reporter sequence.Expression of the reporter sequence is mediated by transcription controlelements derived from a mouse eosinophil peroxidase gene. The method istypically carried out in a transgenic, intact, living rodent. Theanalyte is administered to the transgenic, living rodent. Expression ofthe reporter sequence is monitored wherein an effect on the level ofexpression of the reporter sequence indicates that the analyte affectsexpression mediated by transcription control elements derived from themouse eosinophil peroxidase gene.

Another method relates to monitoring eosinophil cell location in aliving, transgenic rodent. In the method eosinophil production isinduced in the living, transgenic rodent. Eosinophil cell location ismonitored in the living, transgenic rodent by monitoring locations ofexpression of the reporter sequence in regions of the body of theliving, transgenic rodent. The monitoring may be carried out over aseries of time intervals. The monitoring may be begun before, during, orafter inducing eosinophil production.

A further method relates to evaluating the effect of an analyte oneosinophil migration in a living, transgenic rodent. In the method,eosinophil migration is induced at a selected site in first and secondliving, transgenic rodents. An analyte is administered to the firstliving, transgenic rodent. Eosinophil migration to the selected site inthe first and second living, transgenic rodents is monitored bymonitoring expression of the reporter sequence in the living, transgenicrodents. Any effect of the analyte on eosinophil migration in a living,transgenic rodent is evaluated by comparing eosinophil migration in thefirst and second living, transgenic rodents.

Yet another method relates to inducing eosinophil, cell-type specificexpression of a coding sequence of interest in a transgenic mouse. Inthis method eosinophil production is induced in a living, transgenicrodent, wherein the induction of eosinophil production results ineosinophil, cell-type specific expression of the coding sequence ofinterest in the transgenic rodent.

In yet a further aspect, the invention relates to transcription controlelements derived from the genomic locus of the murine eosinophilperoxidase (EPX) gene, wherein the transcription control elements areassociated with a reporter sequence. In particular, recombinant nucleicacid molecules comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:7, aswell as fragments thereof, are described. The invention further relatesto in vivo assay methods that employ animals transfected with suchreporter constructs.

In one aspect, the present invention includes isolated polynucleotidesand/or expression cassettes comprising a polynucleotide having at leastabout 95% identity to the sequence of SEQ ID NO:2, or fragments thereof,operably linked to a coding sequence of interest, wherein thepolynucleotide or fragments thereof comprise at least onetranscriptional control element.

In some embodiments the coding sequence of interest is a reportersequence, for example, a light-generating protein. Such light-generatingproteins comprise bioluminescent proteins (including but not limited to,procaryotic or eucaryotic luciferase) and fluorescent proteins(including but not limited to, blue fluorescent protein, cyanfluorescent protein, green fluorescent protein, yellow fluorescentprotein, and red fluorescent protein, as well as, enhanced and/ordestabilized variants thereof).

The present invention also includes vectors comprising the isolatedpolynucleotides and/or expression cassettes of the present invention.Such vectors typically include a vector backbone, and may be linear orcircular, comprise one or more origins of replication (e.g., a shuttlevector), be site-specifically or randomly integrating, and comprise oneor more selectable or screenable markers.

In one embodiment the present invention includes cells comprising theexpression cassettes and/or vectors of the present invention, e.g.,eosinophil cells and/or precursors thereof. In another embodiment,transgenic, non-human, animals (e.g., rodents, including, but notlimited to, mice, rats, hamsters, gerbils, and guinea pigs) may comprisethe expression cassettes and/or vectors or the present invention. In afurther embodiment, the present invention includes non-human animalsthat comprise a subset of cells comprising the expression cassettesand/or vectors of the present invention, for example, non-human animalsinto which eosinophil cells, or precursors thereof, comprising anexpression cassette of the present invention, have been introduced. Suchnon-human animals may be generated, for example, by administration ofthe eosinophils, comprising expression cassettes and/or vectors of thepresent invention, via intravenous injection.

In yet another aspect, the present invention includes methods of usingthe expression cassettes, vectors, cells, and non-human animals of thepresent invention. In one embodiment, the invention includes a methodfor identifying an analyte capable of modulating expression of a murineeosinophil peroxidase (EPX) gene in a transgenic, living, non-humananimal. Such a method typically comprises administering to the animal ananalyte. The animal comprises one or more of the expression cassettes orvectors of the present invention typically including a reportersequence. Expression of the reporter sequence is monitored. An effect onthe expression of the reporter sequence, which is mediated by theanalyte, indicates that the analyte affects expression of the genecorresponding to the transcriptional control elements which comprise theexpression cassettes and/or vectors employed in the method.

Another method comprises identifying an analyte capable of modulatingexpression of a murine eosinophil peroxidase (EPX) gene in a living,non-human animal. In this method, a mixture comprising eosinophil cells,or precursors thereof, comprising an expression cassette of the presentinvention typically including a reporter sequence, is administered tothe animal concomitant with, before, or after administration of ananalyte. Expression of the reporter sequence is monitored. An effect onthe expression of the reporter sequence, which is mediated by theanalyte, indicates that the analyte affects expression of genecorresponding to the transcriptional control elements which comprise theexpression cassettes and/or vectors employed in the method. In oneembodiment the mixture is administered by intravenous injection.

In a further embodiment of the present invention, the expressioncassettes comprising the transcription control elements of the presentinvention and a reporter, are used to monitor the expression of themurine eosinophil peroxidase (EPX) gene in a cell. In this embodimentexpression of a reporter sequence is monitored in the cell andexpression of the reporter sequence corresponds to expression of genecorresponding to the transcriptional control elements which comprise theexpression cassettes and/or vectors employed in the method.

In yet another aspect of the present invention, the location and/ormigration of labeled eosinophil cells (e.g., carrying an expressioncassette of the present invention comprising a reporter gene) aremonitored in a living, non-human animal. The animal may be a transgenicanimal or an animal into which label cells have been introduced. In oneembodiment, the present invention describes a method for monitoringeosinophil cell location in a living, transgenic animal, e.g., a rodent,by inducing eosinophil production in a living, transgenic rodentcomprising an expression cassette of the present invention. The locationof eosinophil cells are monitored, e.g., temporally and spatially, inthe living, transgenic rodent by monitoring locations of expression ofthe reporter sequence in the living, transgenic rodent. Monitoring ofthe animal may begin before, during, or concurrently with inducement ofeosinophil production.

In a further embodiment, the present invention comprises a method forevaluating the effect of an analyte on eosinophil migration in a living,transgenic rodent. In this method eosinophil migration is induced at aselected site in first and second living, transgenic animals, eachcomprising an expression cassette, comprising a reporter sequence, ofthe present invention. The analyte is administered to the first living,transgenic animal. Eosinophil migration is monitored, as describedherein, to the selected site in the first and second living, transgenicanimals by monitoring expression of the reporter sequence in the living,transgenic animals. Any effect of the analyte on eosinophil migration isevaluated in the living, transgenic animals by comparing eosinophilmigration in the first and second living, transgenic animals.

In a further embodiment of the present invention, a method for inducingeosinophil, cell-type specific expression of a coding sequence ofinterest in a transgenic, non-human animal is described. In this methodeosinophil production is induced in a living, transgenic, non-humananimal, comprising an expression cassette of the present invention,wherein the coding sequence of interest is operably linked to apolynucleotide of the present invention, or fragments thereof, thatcomprise at least one eosinophil, cell-type specific transcriptionalcontrol element. The induction of eosinophil production results ineosinophil, cell-type specific expression of the coding sequence ofinterest in the transgenic animal.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,cell biology and recombinant DNA, which are within the skill of the art.See, e.g., Sambrook, Fritsch, and Maniatis, MOLECULAR CLONING: ALABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULARBIOLOGY, (F. M. Ausubel et al. eds., 1987); the series METHODS INENZYMOLOGY (Academic Press, Inc.); PCR 2: A PRACTICAL APPROACH (M. J.McPherson, B. D. Hames and G. R. Taylor eds., 1995); ANIMAL CELL CULTURE(R. I. Freshney. Ed., 1987); “Transgenic Animal Technology: A LaboratoryHandbook,” by Carl A. Pinkert, (Editor) First Edition, Academic Press;ISBN: 0125571658; and “Manipulating the Mouse Embryo: A LaboratoryManual,” Brigid Hogan, et al., ISBN: 0879693843, Publisher: Cold SpringHarbor Laboratory Press, Pub. Date: September 1999, Second Edition.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

1. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below. Unlessotherwise indicated, all terms used herein have the same meaning as theywould to one skilled in the art of the present invention.

The terms “nucleic acid molecule” and “polynucleotide” are usedinterchangeably to and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Polynucleotides may have any three-dimensional structure, andmay perform any function, known or unknown. Non-limiting examples ofpolynucleotides include a gene, a gene fragment, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers.

A polynucleotide is typically composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus,the term polynucleotide sequence is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching.

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide,for example, in vivo when placed under the control of appropriateregulatory sequences (or “control elements”). The boundaries of thecoding sequence are typically determined by a start codon at the 5′(amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. A coding sequence can include, but is not limited to, cDNAfrom viral, procaryotic or eucaryotic mRNA, genomic DNA sequences fromviral or procaryotic DNA, and even synthetic DNA sequences. Atranscription termination sequence may be located 3′ to the codingsequence. Other “control elements” may also be associated with a codingsequence. A DNA sequence encoding a polypeptide can be optimized forexpression in a selected cell by using the codons preferred by theselected cell to represent the DNA copy of the desired polypeptidecoding sequence. “Encoded by” refers to a nucleic acid sequence whichcodes for a polypeptide sequence, wherein the polypeptide sequence or aportion thereof contains an amino acid sequence of at least 3 to 5 aminoacids, more preferably at least 8 to 10 amino acids, and even morepreferably at least 15 to 20 amino acids from a polypeptide encoded bythe nucleic acid sequence. Also encompassed are polypeptide sequences,which are immunologically identifiable with a polypeptide encoded by thesequence.

A “transcription factor” typically refers to a protein (or polypeptide)which affects the transcription, and accordingly the expression, of aspecified gene. A transcription factor may refer to a single polypeptidetranscription factor, one or more polypeptides acting sequentially or inconcert, or a complex of polypeptides.

Typical “control elements” include, but are not limited to,transcription promoters, transcription enhancer elements, cis-actingtranscription regulating elements (transcription regulators, e.g., acis-acting element that affects the transcription of a gene, forexample, a region of a promoter with which a transcription factorinteracts to induce or repress expression of a gene), transcriptioninitiation signals (e.g., TATA box), basal promoters, transcriptiontermination signals, as well as polyadenylation sequences (located 3′ tothe translation stop codon), sequences for optimization of initiation oftranslation (located 5′ to the coding sequence), translation enhancingsequences, and translation termination sequences. Transcriptionpromoters can include, for example, inducible promoters (whereexpression of a polynucleotide sequence operably linked to the promoteris induced by an analyte, cofactor, regulatory protein, etc.),repressible promoters (where expression of a polynucleotide sequenceoperably linked to the promoter is induced by an analyte, cofactor,regulatory protein, etc.), and constitutive promoters.

“Expression enhancing sequences,” also referred to as “enhancersequences” or “enhancers,” typically refer to control elements thatimprove transcription or translation of a polynucleotide relative to theexpression level in the absence of such control elements (for example,promoters, promoter enhancers, enhancer elements, and translationalenhancers (e.g., Shine and Delagarno sequences)).

The term “modulation” refers to both inhibition, including partialinhibition, as well as stimulation. Thus, for example, a compound thatmodulates expression of a reporter sequence may either inhibit thatexpression, either partially or completely, or stimulate expression ofthe sequence.

“Purified polynucleotide” refers to a polynucleotide of interest orfragment thereof which is essentially free, e.g., contains less thanabout 50%, preferably less than about 70%, and more preferably less thanabout 90%, of the protein with which the polynucleotide is naturallyassociated. Techniques for purifying polynucleotides of interest arewell known in the art and include, for example, disruption of the cellcontaining the polynucleotide with a chaotropic agent and separation ofthe polynucleotide(s) and proteins by ion-exchange chromatography,affinity chromatography and sedimentation according to density.

A “heterologous sequence” typically refers to either (i) a nucleic acidsequence that is not normally found in the cell or organism of interest,or (ii) a nucleic acid sequence introduced at a genomic site wherein thenucleic acid sequence does not normally occur in nature at that site.For example, a DNA sequence encoding a polypeptide can be obtained fromyeast and introduced into a bacterial cell. In this case the yeast DNAsequence is “heterologous” to the native DNA of the bacterial cell.Alternatively, a promoter sequence, for example, from a Tie2 gene can beintroduced into the genomic location of a fosB gene. In this case theTie2 promoter sequence is “heterologous” to the native fosB genomicsequence.

A “polypeptide” is used in it broadest sense to refer to a compound oftwo or more subunit amino acids, amino acid analogs, or otherpeptidomimetics. The subunits may be linked by peptide bonds or by otherbonds, for example ester, ether, etc. The term “amino acid” refers toeither natural and/or unnatural or synthetic amino acids, includingglycine and both the D or L optical isomers, and amino acid analogs andpeptidomimetics. A peptide of three or more amino acids is commonlycalled an oligopeptide if the peptide chain is short. If the peptidechain is long, the peptide is typically called a polypeptide or aprotein.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given promoter that is operably linked to a codingsequence (e.g., a reporter expression cassette) is capable of effectingthe expression of the coding sequence when the proper enzymes arepresent. The promoter or other control elements need not be contiguouswith the coding sequence, so long as they function to direct theexpression thereof. For example, intervening untranslated yettranscribed sequences can be present between the promoter sequence andthe coding sequence and the promoter sequence can still be considered“operably linked” to the coding sequence.

“Recombinant” describes a nucleic acid molecule means a polynucleotideof genomic, cDNA, semisynthetic, or synthetic origin which, by virtue ofits origin or manipulation: (1) is not associated with all or a portionof the polynucleotide with which it is associated in nature; and/or (2)is linked to a polynucleotide other than that to which it is linked innature. The term “recombinant” as used with respect to a protein orpolypeptide means a polypeptide produced by expression of a recombinantpolynucleotide. “Recombinant host cells,” “host cells,” “cells,” “celllines,” “cell cultures,” and other such terms denoting procaryoticmicroorganisms or eucaryotic cell lines cultured as unicellularentities, are used interchangeably, and refer to cells which can be, orhave been, used as recipients for recombinant vectors or other transferDNA, and include the progeny of the original cell which has beentransfected. It is understood that the progeny of a single parental cellmay not necessarily be completely identical in morphology or in genomicor total DNA complement to the original parent, due to accidental ordeliberate mutation. Progeny of the parental cell which are sufficientlysimilar to the parent to be characterized by the relevant property, suchas the presence of a nucleotide sequence encoding a desired peptide, areincluded in the progeny intended by this definition, and are covered bythe above terms.

An “isolated polynucleotide” molecule is a nucleic acid moleculeseparate and discrete from the whole organism with which the molecule isfound in nature; or a nucleic acid molecule devoid, in whole or part, ofsequences normally associated with it in nature; or a sequence, as itexists in nature, but having heterologous sequences (as defined below)in association therewith.

Techniques for determining nucleic acid and amino acid “sequenceidentity” also are known in the art. Typically, such techniques includedetermining the nucleotide sequence of the mRNA for a gene and/ordetermining the amino acid sequence encoded thereby, and comparing thesesequences to a second nucleotide or amino acid sequence. In general,“identity” refers to an exact nucleotide-to-nucleotide or aminoacid-to-amino acid correspondence of two polynucleotides or polypeptidesequences, respectively. Two or more sequences (polynucleotide or aminoacid) can be compared by determining their “percent identity.” Thepercent identity of two sequences, whether nucleic acid or amino acidsequences, is the number of exact matches between two aligned sequencesdivided by the length of the shorter sequences and multiplied by 100. Anapproximate alignment for nucleic acid sequences is provided by thelocal homology algorithm of Smith and Waterman, Advances in AppliedMathematics 2:482-489 (1981). This algorithm can be applied to aminoacid sequences by using the scoring matrix developed by Dayhoff, Atlasof Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl.3:353-358, National Biomedical Research Foundation, Washington, D.C.,USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763(1986). An exemplary implementation of this algorithm to determinepercent identity of a sequence is provided by the Genetics ComputerGroup (Madison, Wis.) in the “BestFit” utility application. The defaultparameters for this method are described in the Wisconsin SequenceAnalysis Package Program Manual, Version 8 (1995) (available fromGenetics Computer Group, Madison, Wis.). A preferred method ofestablishing percent identity in the context of the present invention isto use the MPSRCH package of programs copyrighted by the University ofEdinburgh, developed by John F. Collins and Shane S. Sturrok, anddistributed by IntelliGenetics, Inc. (Mountain View, Calif.). From thissuite of packages the Smith-Waterman algorithm can be employed wheredefault parameters are used for the scoring table (for example, gap openpenalty of 12, gap extension penalty of one, and a gap of six). From thedata generated the “Match” value reflects “sequence identity.” Othersuitable programs for calculating the percent identity or similaritybetween sequences are generally known in the art, for example, anotheralignment program is BLAST, used with default parameters. For example,BLASTN and BLASTP can be used using the following default parameters:genetic code=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+Swiss protein+Spupdate+PIR.

One of skill in the art can readily determine the proper searchparameters to use for a given sequence in the above programs. Forexample, the search parameters may vary based on the size of thesequence in question. Thus, for example, a representative embodiment ofthe present invention would include an isolated polynucleotidecomprising X contiguous nucleotides, wherein (i) the X contiguousnucleotides have at least about 50% identity to Y contiguous nucleotidesderived from any of the sequences described herein, (ii) X equals Y, and(iii) X is equal to from 6 up to the number of nucleotides present in aselected full-length sequence as described herein (e.g., see theExamples, Figures, Sequence Listing and claims), including all integervalues falling within the above-described ranges. A “fragment” of apolynucleotide refers to any length polynucleotide molecule derived froma larger polynucleotide described herein (i.e., Y contiguousnucleotides, where X=Y as just described). Exemplary fragment lengthsinclude, but are not limited to, at least about 6 contiguousnucleotides, at least about 50 contiguous nucleotides, about 100contiguous nucleotides, about 250 contiguous nucleotides, about 500contiguous nucleotides, or at least about 1000 contiguous nucleotides ormore, wherein such contiguous nucleotides are derived from a largersequence of contiguous nucleotides.

The purified polynucleotides and polynucleotides used in construction ofexpression cassettes of the present invention include the sequencesdisclosed herein as well as related polynucleotide sequences havingsequence identity of approximately 80% to 100% and integer valuestherebetween. Typically the percent identities between the sequencesdisclosed herein and the claimed sequences are at least about 80-85%,preferably at least about 90-92%, more preferably at least about 95%,and most preferably at least about 98% sequence identity (including allinteger values falling within these described ranges). These percentidentities are, for example, relative to the claimed sequences, or othersequences of the present invention, when the sequences of the presentinvention are used as the query sequence.

Alternatively, the degree of sequence similarity between polynucleotidescan be determined by hybridization of polynucleotides under conditionsthat form stable duplexes between homologous regions, followed bydigestion with single-stranded-specific nuclease(s), and sizedetermination of the digested fragments. Two DNA, or two polypeptidesequences are “substantially homologous” to each other when thesequences exhibit at least about 80-85%, preferably 85-90%, morepreferably 90-95%, and most preferably 98-100% sequence identity to thereference sequence over a defined length of the molecules, as determinedusing the methods above. Substantially homologous also refers tosequences showing complete identity to the specified DNA or polypeptidesequence. DNA sequences that are substantially homologous can beidentified in a Southern hybridization experiment under, for example,stringent conditions, as defined for that particular system. Definingappropriate hybridization conditions is within the skill of the art.See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic AcidHybridization, supra.

Two nucleic acid fragments are considered to “selectively hybridize” asdescribed herein. The degree of sequence identity between two nucleicacid molecules affects the efficiency and strength of hybridizationevents between such molecules. A partially identical nucleic acidsequence will at least partially inhibit a completely identical sequencefrom hybridizing to a target molecule. Inhibition of hybridization ofthe completely identical sequence can be assessed using hybridizationassays that are well known in the art (e.g., Southern blot, Northernblot, solution hybridization, or the like, see Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N.Y.). Such assays can be conducted using varying degreesof selectivity, for example, using conditions varying from low to highstringency. If conditions of low stringency are employed, the absence ofnon-specific binding can be assessed using a secondary probe that lackseven a partial degree of sequence identity (for example, a probe havingless than about 30% sequence identity with the target molecule), suchthat, in the absence of non-specific binding events, the secondary probewill not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a target nucleic acid sequence,and then by selection of appropriate conditions the probe and the targetsequence “selectively hybridize,” or bind, to each other to form ahybrid molecule. A nucleic acid molecule that is capable of hybridizingselectively to a target sequence under “moderately stringent” typicallyhybridizes under conditions that allow detection of a target nucleicacid sequence of at least about 10-14 nucleotides in length having atleast approximately 70% sequence identity with the sequence of theselected nucleic acid probe. Stringent hybridization conditionstypically allow detection of target nucleic acid sequences of at leastabout 10-14 nucleotides in length having a sequence identity of greaterthan about 90-95% with the sequence of the selected nucleic acid probe.Hybridization conditions useful for probe/target hybridization where theprobe and target have a specific degree of sequence identity, can bedetermined as is known in the art (see, for example, Nucleic AcidHybridization: A Practical Approach, editors B. D. Hames and S. J.Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of probe and target sequences, basecomposition of the various sequences, concentrations of salts and otherhybridization solution components, the presence or absence of blockingagents in the hybridization solutions (e.g., formamide, dextran sulfate,and polyethylene glycol), hybridization reaction temperature and timeparameters, as well as, varying wash conditions. The selection of aparticular set of hybridization conditions is selected followingstandard methods in the art (see, for example, Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N.Y.).

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells. Thus, the term includes cloning, and expressionvehicles, as well as integrating vectors.

“Nucleic acid expression vector” or “expression cassette” refers to anassembly that is capable of directing the expression of a sequence orgene of interest. The nucleic acid expression vector includes a promoterthat is operably linked to the sequences or gene(s) of interest. Othercontrol elements may be present as well. Expression cassettes describedherein may be contained within a plasmid construct. In addition to thecomponents of the expression cassette, the plasmid construct may alsoinclude a bacterial origin of replication, one or more selectablemarkers, a signal which allows the plasmid construct to exist assingle-stranded DNA (e.g., a M13 origin of replication), a multiplecloning site, and a “mammalian” origin of replication (e.g., a SV40 oradenovirus origin of replication).

An “expression cassette” comprises any nucleic acid construct capable ofdirecting the expression of a gene/coding sequence of interest. Suchcassettes can be constructed into a “vector,” “vector construct,”“expression vector,” or “gene transfer vector,” in order to transfer theexpression cassette into target cells. Thus, the term includes cloningand expression vehicles, as well as viral vectors.

A variety of “reporter genes” also referred to as “reporter sequences”and “marker sequences,” i.e., genes or sequences the expression of whichindicates the expression of polynucleotide sequences of interest towhich the reporter gene or sequence is operably linked. Preferred arethose reporter sequences that produce a protein product that is easilymeasured, preferably in a routine assay. Suitable reporter genesinclude, but are not limited to chloramphenicol acetyl transferase(CAT), light generating proteins (e.g., luc-encoded, lux-encoded,fluorescent proteins), and beta-galactosidase. Convenient assaysinclude, but are not limited to calorimetric, fluorimetric and enzymaticassays. In one aspect, reporter genes may be employed that are expressedwithin the cell and whose extracellular products are directly measuredin the intracellular medium, or in an extract of the intracellularmedium of a cultured cell line. This provides advantages over using areporter gene whose product is secreted, since the rate and efficiencyof the secretion introduces additional variables that may complicateinterpretation of the assay. In a preferred embodiment, the reportergene is a light generating protein. When using the light generatingreporter proteins described herein, expression can be evaluatedaccurately and non-invasively as described above (see, for example,Contag, P. R., et al., (1998) Nature Med. 4:245-7; Contag, C. H., etal., (1997) Photochem Photobiol. 66:523-31; Contag, C. H., et al.,(1995) Mol Microbiol. 18:593-603).

A “light generating protein” or “light-emitting protein” is abioluminescent or fluorescent protein capable of producing lighttypically in the range of 200 nm to 1100 nm, preferably in the visiblespectrum (i.e., between approximately 350 nm and 800 nm). Bioluminescentproteins produce light through a chemical reaction (typically requiringa substrate, energy source, and oxygen). Fluorescent proteins producelight through the absorption and re-emission of radiation (such as withgreen fluorescent protein). Examples of bioluminescent proteins include,but are not limited to, the following: “luciferase,” unless statedotherwise, includes procaryotic (e.g., bacterial lux-encoded) andeucaryotic (e.g., firefly luc-encoded) luciferases, as well as variantspossessing varied or altered optical properties, such as luciferasesthat produce different colors of light (e.g., Kajiyama, N., and Nakano,E., Protein Engineering 4(6):691-693 (1991)); and “photoproteins,” forexample, calcium activated photoproteins (e.g., Lewis, J. C., et al.,Fresenius J. Anal Chem. 366(6-7):760-768 (2000)). Examples offluorescent proteins include, but are not limited to, green, yellow,cyan, blue, and red fluorescent proteins (e.g., Hadjantonakis, A. K., etal., Histochem. Cell Biol. 115(1):49-58 (2001)).

“Bioluminescent protein substrate” describes a substrate of alight-generating protein, e.g., luciferase enzyme, that generates anenergetically decayed substrate (e.g., luciferin) and a photon of lighttypically with the addition of an energy source, such as ATP or FMNH2,and oxygen. Examples of such substrates include, but are not limited to,decanal in the bacterial lux system,4,5-dihydro-2-(6-hydroxy-2-benzothiazolyl)-4-thiazolecarboxylic acid (orsimply called luciferin) in the Firefly luciferase (luc) system, “panal”in the bioluminescent fungus Panellus stipticus system (Tetrahedron44:1597-1602, 1988) and N-iso-valeryl-3-aminopropanol in the earth wormDiplocardia longa system (Biochem. 15:1001-1004, 1976). In some systems,as described herein, aldehyde can be used as a substrate for thelight-generating protein.

“Light” is defined herein, unless stated otherwise, as electromagneticradiation having a wavelength of between about 200 nm (e.g., for UV-C)and about 1100 nm (e.g., infrared). The wavelength of visible lightranges between approximately 350 nm to approximately 800 nm (i.e.,between about 3,500 angstroms and about 8,000 angstroms).

“Animal” typically refers to a non-human animal, including, withoutlimitation, farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingferrets, hares and rabbits, rodents, such as mice, rats, hamsters,gerbils, and guinea pigs; non-human primates, including chimpanzees. Theterm “animal” may also include, without limitation; birds, includingdomestic, wild and game birds such as chickens, turkeys and othergallinaceous birds, ducks, geese, and the like, as well as amphibians,fish, insects, reptiles, etc. The term does not denote a particular age.Thus, adult, embryonic, fetal, and newborn individuals are intended tobe covered.

A “transgenic animal” refers to a genetically engineered animal oroffspring of genetically engineered animals. A transgenic animal usuallycontains material from at least one unrelated organism, such as from avirus, microorganism, plant, or other animal. The term “chimeric animal”is used to refer to animals in which the heterologous gene is found, orin which the heterologous gene is expressed in some but not all cells ofthe animal.

“Analyte” refers to any compound or substance whose effects (e.g.,induction or repression of a specific promoter) can be evaluated usingthe test animals and methods of the present invention. Such analytesinclude, but are not limited to, chemical compounds, pharmaceuticalcompounds, polypeptides, peptides, polynucleotides, and polynucleotideanalogs. Many organizations (e.g., the National Institutes of Health,pharmaceutical and chemical corporations) have large libraries ofchemical or biological compounds from natural or synthetic processes, orfermentation broths or extracts. Such compounds/analytes can be employedin the practice of the present invention.

The term “positive selection marker” refers to a gene encoding a productthat enables only the cells that carry the gene to survive and/or growunder certain conditions. For example, plant and animal cells thatexpress the introduced neomycin resistance (Neo^(r)) gene are resistantto the compound G418. Cells that do not carry the Neo^(r) gene markerare killed by G418. Other positive selection markers will be known tothose of skill in the art. Typically, positive selection markers encodeproducts that can be readily assayed. Thus, positive selection markerscan be used to determine whether a particular DNA construct has beenintroduced into a cell, organ or tissue.

“Negative selection marker” refers to gene encoding a product that canbe used to selectively kill and/or inhibit growth of cells under certainconditions. Non-limiting examples of negative selection inserts includea herpes simplex virus (HSV)-thymidine kinase (TK) gene. Cellscontaining an active HSV-TK gene are incapable of growing in thepresence of gangcylovir or similar agents. Thus, depending on thesubstrate, some gene products can act as either positive or negativeselection markers.

The term “homologous recombination” refers to the exchange of DNAfragments between two DNA molecules or chromatids at the site ofessentially identical nucleotide sequences. It is understood thatsubstantially homologous sequences can accommodate insertions,deletions, and substitutions in the nucleotide sequence. Thus, linearsequences of nucleotides can be essentially identical even if some ofthe nucleotide residues do not precisely correspond or align (see,above).

A “knock-out” mutation refers to partial or complete loss of expressionof at least a portion the target gene. Examples of knock-out mutationsinclude, but are not limited to, gene-replacement by heterologoussequences, gene disruption by heterologous sequences, and deletion ofessential elements of the gene (e.g., promoter region, portions of acoding sequence). A “knock-out” mutation is typically identified by thephenotype generated by the mutation.

A “gene” as used in the context of the present invention is a sequenceof nucleotides in a genetic nucleic acid (chromosome, plasmid, etc.)with which a genetic function is associated. A gene is a hereditaryunit, for example of an organism, comprising a polynucleotide sequence(e.g., a DNA sequence for mammals) that occupies a specific physicallocation (a “locus”, “gene locus” or “genetic locus”) within the genomeof an organism. A gene can encode an expressed product, such as apolypeptide or a polynucleotide (e.g., tRNA). Alternatively, a gene maydefine a genomic location for a particular event/function, such as thebinding of proteins and/or nucleic acids (e.g., phage attachment sites),wherein the gene does not encode an expressed product. Typically, a geneincludes coding sequences, such as, polypeptide encoding sequences, andnon-coding sequences, such as, transcription control elements (e.g.,promoter sequences), polyadenlyation sequences, transcriptionalregulatory sequences (e.g., enhancer sequences). Many eucaryotic geneshave “exons” (coding sequences) interrupted by “introns” (non-codingsequences). In certain cases, a gene may share sequences with anothergene(s) (e.g., overlapping genes).

The “native sequence” or “wild-type sequence” of a gene is thepolynucleotide sequence that comprises the genetic locus correspondingto the gene, e.g., all regulatory and open-reading frame codingsequences required for expression of a completely functional geneproduct as they are present in the wild-type genome of an organism. Thenative sequence of a gene can include, for example, transcriptionalpromoter sequences, translation enhancing sequences, introns, exons, andpoly-A processing signal sites. It is noted that in the generalpopulation, wild-type genes may include multiple prevalent versions thatcontain alterations in sequence relative to each other and yet do notcause a discernible pathological effect. These variations are designated“polymorphisms” or “allelic variations.”

By “replacement sequence” is meant a polynucleotide sequence that issubstituted for at least a portion of the native or wild-type sequenceof a gene.

“Linear vector” or “linearized vector,” is a vector having two ends. Forexample, circular vectors, such as plasmids, can be linearized bydigestion with a restriction endonuclease that cuts at a single site inthe plasmid. Preferably, the expression vectors described herein arelinearized such that the ends are not within the sequences of interest.

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor method parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of methods and materials similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

2. Modes of Carrying out the Invention

Throughout this application, various publications, patents, andpublished patent applications are referred to by an identifyingcitation. The disclosures of these publications, patents, and publishedpatent specifications referenced in this application are herebyincorporated by reference into the present disclosure to more fullydescribe the state of the art to which this invention pertains.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise. Thus, for example, reference to “anexpression construct” includes a mixture of two or more such agents.

2.1 General Overview

In one aspect, the present invention relates to novel transcriptioncontrol elements derived from the mouse eosinophil peroxidase (EPX) genelocus, expression cassettes which include those control elements, vectorconstructs, cells and transgenic animals containing the expressioncassettes, and methods of using the cells and transgenic animalscontaining the expression cassettes, for example, as modeling, screeningand/or test systems. Methods of using the control elements, expressioncassettes, cells, and transgenic animals of the present inventioninclude, but are not limited to, studies involving host immunity,allergic reactions and drug metabolism, and methods for screening forcompounds which affect expression of the mouse eosinophil peroxidase(EPX) gene. Exemplary transcription control elements useful in thepractice of the present invention include those derived from mouseeosinophil peroxidase (EPX) gene locus.

In one embodiment, the present invention relates to (1) noveltranscription control elements (e.g., promoters) derived from the mouseeosinophil peroxidase (EPX) gene locus; (2) expression cassettescomprising such transcription control elements operatively linked togenes encoding a gene product, such as, a reporter, a protein,polypeptide, hormone, ribozyme, or antisense RNA, (3) recombinant cellscomprising such expression cassettes, (4) methods of screening usingsuch cells (e.g., screening for the effects of a compound or compoundson expression of the mouse eosinophil peroxidase (EPX) gene, (5) animals(e.g., transgenic or transiently transfected) comprising theaforementioned novel transcription control elements, expressioncassettes and vector constructs, (6) methods of monitoring the effect ofa compound or compounds on expression of the mouse eosinophil peroxidase(EPX) using such animals, and (7) methods of evaluating pathophysiologyinvolving the mouse eosinophil peroxidase (EPX) using such animals.

Non-invasive imaging and/or detecting of light-emitting conjugates inmammalian subjects was described in U.S. Pat. Nos. 5,650,135, and6,217,847, by Contag, et al., issued 22 Jul. 1997, and Apr. 17, 2001,respectively, and herein incorporated by reference. This imagingtechnology can be used in the practice of the present invention in viewof the teachings of the present specification. In the imaging method,the conjugates contain a biocompatible entity and a light-generatingmoiety. Biocompatible entities include, but are not limited to, smallmolecules such as cyclic organic molecules; macromolecules such asproteins; microorganisms such as viruses, bacteria, yeast and fungi;eucaryotic cells; all types of pathogens and pathogenic substances; andparticles such as beads and liposomes. In another aspect, biocompatibleentities may be all or some of the cells that constitute the mammaliansubject being imaged, for example, cells carrying the expressioncassettes of the present invention expressing a reporter sequence.

Light-emitting capability is conferred on the biocompatible entities bythe conjugation of a light-generating moiety. Such moieties includefluorescent molecules, fluorescent proteins, enzymatic reactions whichgive off photons, and luminescent substances, such as bioluminescentproteins. In the context of the present invention, light emittingcapability is typically conferred on target cells by having at least onecopy of a light-generating protein, e.g., a luciferase, present. Inpreferred embodiments, luciferase is operably linked to appropriatecontrol elements that can facilitate expression of a polypeptide havingluciferase activity. Substrates of luciferase can be endogenous to thecell or applied to the cell or system (e.g., injection into a transgenicmouse, having cells carrying a luciferase construct, of a suitablesubstrate for the luciferase, for example, luciferin). The conjugationmay involve a chemical coupling step, genetic engineering of a fusionprotein, or the transformation of a cell, microorganism or animal toexpress a light-generating protein.

Thus, in one aspect, the present invention relates to animal testsystems and methods for studies of an analyte of interest for theireffects on expression of the mouse eosinophil peroxidase (EPX) gene. Inthe practice of the present invention, transgenic mammals (e.g.,rodents, including, but not limited to, mice or rats) are constructedwhere control elements, for example, a promoter or transcriptionalregulatory sequence from the mouse eosinophil peroxidase (EPX) genelocus are operably linked to reporter gene coding sequences (forexample, luciferase). An appropriate substrate for the reporter geneproduct is administered to the animal in addition to an analyte ofinterest. The order of administration of these two substances can beempirically determined for each analyte of interest. Induction ofexpression mediated by any of the control elements is then evaluated bynon-invasive imaging methods using the whole animal.

Thus, in one aspect of the present invention, animals described hereincan be used to evaluate the in vivo effects of therapeutic substances onthe expression of the mouse eosinophil peroxidase (EPX) gene. Asdescribed above, eosinophils play a protective role in host immunity toparasitic worm infections and, detrimentally, are involved in thepathophysiology of asthma and other allergic diseases. Eosinophils areprominent in airway inflammation. Eosinophils are involved in diseaselike asthma, chronic eosinophilic pneumonia, Churg-Strauss Syndrome,Hypereosinophilic syndrome, allergic rhinitis, atopic dermatitis. Thepresent invention allows marking eosinophils in vivo. Thus an eosinophilmigration model can be established in animals, e.g., transgenic rodents,so that the effects of drug candidates can be tested efficiently. Theeosinophil peroxidase (EPX) gene is known to be exclusively expressed ineosinophils. Accordingly, linking transcription control elements,associated with the expression of this gene, to a reporter sequenceallows the specific tagging (or marking) of eosinophils in vivo.

2.2 Promoters

The expression cassettes, vectors, cells and transgenic animalsdescribed herein contain a sequence encoding a detectable gene product,e.g., a luciferase gene, operably linked to a transcription controlelement, e.g., a promoter. The promoter may be from the same species asthe transgenic animal (e.g., mouse promoter used in construct to maketransgenic mouse) or from a different species (e.g., mouse promoter usedin construct to make transgenic rat). In one embodiment of the presentinvention, the promoter is derived from the mouse eosinophil peroxidase(EPX) gene. Based on the teachings of the present invention, eosinophilperoxidase (EPX) genes may be isolated from other sources as well, e.g.,human, rat, or guinea pig. Thus, when a drug is administered to atransgenic animal carrying a vector construct of the present invention,the promoter may be induced or repressed and expression of the reporter,e.g., luciferase, can then be monitored in vivo.

Exemplary transcription control elements (e.g., promoters) for use inthe present invention include, but are not limited to, promoters derivedfrom eosinophil peroxidase (EPX) genes. Exemplified herein are noveltranscription control elements derived from the genomic locus of themouse eosinophil peroxidase (EPX) gene.

As one of skill in the art will appreciate in view of the teachings ofthe present specification, transcription control element sequences canbe derived and isolated from, e.g., genomic sequences, using methodknown in the art in view of the teachings herein. For example, thetranscription control element sequences of the mouse eosinophilperoxidase (EPX) gene were isolated and sequenced as described inExample 1 below.

Another exemplary method of isolating promoter sequences employs aGenomeWalker® kit, commercially available from Clontech (Palo Alto,Calif.), and described on page 27 of the 1997-1998 Clontech catalog.

The subject nucleic acids of the present invention (e.g., as describedin Example 1) find a wide variety of applications including use ashybridization probes, PCR primers, expression cassettes useful forcompound screening, detecting the presence of the mouse eosinophilperoxidase (EPX) gene or variants thereof, detecting the presence ofgene transcripts, detecting or amplifying nucleic acids encodingadditional eosinophil peroxidase (EPX) gene promoter sequences orhomologues thereof (as well as, structural analogs), and in a variety ofscreening assays.

A wide variety of assays for transcriptional expression can be usedbased on the teaching of the present specification, including, but notlimited to, cell-based transcription assays, screening in vivo intransgenic animals, and promoter-protein binding assays. For example,the disclosed luciferase reporter constructs are used to transfecteosinophil cells for cell-based transcription assays. For example,eosinophil cells are plated onto microtiter plates and used to screenlibraries of candidate agents for compounds which modulate thetranscriptional regulation of the eosinophil peroxidase (EPX) genepromoter, as monitored by luciferase expression.

Experiments performed in support of the present invention involve theisolation and sequencing of a transcriptional control elements of amouse eosinophil peroxidase (EPX) gene, generation of transgenic micecomprising these transcriptional control elements operatively linked tosequences encoding a light-generating protein (e.g., luciferase), andthe utilization of these transgenic mice to establish an eosinophilmigration model. The mouse eosinophil peroxidase (EPX) gene promotersequences have not been previously characterized. Identification of thesequence of the transcriptional control regions of the mouse eosinophilperoxidase (EPX) gene will facilitate the analysis of eosinophilperoxidase (EPX) gene expression regulation studies in vitro and invivo. An animal, e.g., mouse, eosinophil migration model will greatlyenhance the process of validating compounds (or analytes) that can beused in the management of eosinophil-related disease states, that is,disease states where eosinophils serve as a marker.

As noted above, the present invention relates to a recombinant nucleicacid molecule comprising transcription control elements derived from amouse eosinophil peroxidase (EPX) gene locus. Isolation andcharacterization of these sequences is described below in Example 1. Inparticular, recombinant nucleic acid molecules comprising SEQ ID NO:1and SEQ ID NO:2, as well as fragments thereof, are described. Thefragments have approximately 80% to 100%, and integer valuestherebetween, sequence identity to sequences disclosed, at least 80-85%,preferably 85-90%, more preferably 90-95%, and most preferably 98-100%sequence identity to the reference sequence (i.e., the sequences of thepresent invention). The present invention may also include a nucleicacid sequence substantially complementary to said polynucleotidesequences, or fragments thereof, as well as, a nucleic acid sequencethat specifically hybridizes to said polynucleotide sequences orfragments thereof.

The invention includes further transcription control element sequences(e.g., promoter sequences) identified based on the teachings of thepresent specification (including, but not limited to, sequenceinformation and isolation methods, e.g., Example 1).

The nucleic acid molecules of this invention are useful for producingtransfected cells and transgenic animals that are themselves useful in avariety of applications, for example, for screening for compounds thataffect transcription mediated by the mouse eosinophil peroxidase (EPX)gene transcriptional control elements.

Those skilled in the art can practice the invention by following theguidance of the specification supplemented with standard procedures ofmolecular biology for the isolation and characterization of mouseeosinophil peroxidase (EPX) gene locus transcription control elements,their transfection into host cells, and expression of heterologous DNAoperably linked to said EPX promoters. For example, DNA is commonlytransferred or introduced into recipient mammal cells by calciumphosphate-mediated gene transfer, electroporation, lipofection, viralinfection, and the like. General methods and vectors for gene transferand expression may be found, for example, in M. Kriegler, Gene Transferand Expression: A Laboratory Manual, Stockton Press (1990). Direct genetransfer to cells in vivo can be achieved, for example, by the use ofmodified viral vectors, including, but not limited to, retroviruses,adenoviruses, adeno-associated viruses and herpes viruses, liposomes,and direct injection of DNA into certain cell types. In this manner,recombinant expression vectors and recombinant cells containing thenovel EPX transcription control elements of the present inventionoperably linked to a desired heterologous gene can be delivered tospecific target cells in vivo. See, e.g., Wilson, Nature, 365: 691-692(1993); Plautz et al, Annals NY Acad. Sci., 716: 144-153 (1994); Farhoodet al, Annals NY Acad. Sci., 716: 23-34 (1994) and Hyde et al Nature,362: 250-255 (1993). Furthermore, cells may be transformed ex vivo andintroduced directly at localized sites by injection, e.g.,intra-articular, intracutaneous, intramuscular and the like.

Cloning and characterization of the EPX-locus-derived transcriptioncontrol elements are described in Example 1, below.

Activity of the transcription control element sequences comprising theexpression cassettes and vectors of the present invention may bemonitored by detecting and/or quantifying the protein products encodedby the reporter sequences operably linked to those promoters. Theparticular method used to monitor promoter activity depends on thereporter sequence employed, and may include, for example, enzymaticassay methods, as well as, in the case of reporter sequences whichencode light-generating proteins, in vitro or in vivo bioluminescentimaging.

Monitoring promoter activity in turn enables one to monitor thebiological processes with which that promoter is associated. It mayfurther be employed in methods of screening analytes which modulatethose processes at the promoter level (see below).

2.3 Expression Cassettes and Vectors

The expression cassettes described herein may typically include thefollowing components: (1) a polynucleotide encoding a reporter gene,such as a sequence encoding a light generating protein, (2) atranscription control element operably linked to the reporter genesequence, wherein the control element is heterologous to the codingsequences of the light generating protein (e.g., the novel EPX sequencesof the present invention). Transcription control elements derived fromthe sequences provided herein may be associated with, for example, abasal transcription promoter to confer regulation provided by suchcontrol elements on such a basal transcription promoter. Exemplaryexpression constructs are described in Example 1.

The present invention also includes providing such expression cassettesin vectors, comprising, for example, a suitable vector backbone andoptionally a sequence encoding a selection marker e.g., a positive ornegative selection marker. Suitable vector backbones generally includean F1 origin of replication; a colE1 plasmid-derived origin ofreplication; polyadenylation sequence(s); sequences encoding antibioticresistance (e.g., ampicillin resistance) and other regulatory or controlelements. Non-limiting examples of appropriate backbones include:pBluescriptSK (Stratagene, La Jolla, Calif.); pBluescriptKS (Stratagene,La Jolla, Calif.) and other commercially available vectors.

A variety of reporter genes may be used in the practice of the presentinvention. Preferred are those that produce a protein product which iseasily measured in a routine assay. Suitable reporter genes include, butare not limited to chloramphenicol acetyl transferase (CAT), lightgenerating proteins (e.g., luciferase), and beta-galactosidase.Convenient assays include, but are not limited to calorimetric,fluorimetric and enzymatic assays. In one aspect, reporter genes may beemployed that are expressed within the cell and whose extracellularproducts are directly measured in the intracellular medium, or in anextract of the intracellular medium of a cultured cell line. Thisprovides advantages over using a reporter gene whose product issecreted, since the rate and efficiency of the secretion introducesadditional variables that may complicate interpretation of the assay. Ina preferred embodiment, the reporter gene is a light generating protein.When using the light generating reporter proteins described herein,expression can be evaluated accurately and non-invasively as describedabove (see, for example, Contag, P. R., et al., (1998) Nature Med.4:245-7; Contag, C. H., et al., (1997) Photochem Photobiol. 66:523-31;Contag, C. H., et al., (1995) Mol Microbiol. 18:593-603).

In one aspect of the invention, the light generating is luciferase.Luciferase coding sequences useful in the practice of the presentinvention include sequences obtained from lux genes (procaryotic genesencoding a luciferase activity) and luc genes (eucaryotic genes encodinga luciferase activity). A variety of luciferase encoding genes have beenidentified including, but not limited to, the following: B. A. Sherf andK. V. Wood, U.S. Pat. No. 5,670,356, issued 23 Sep. 1997; Kazami, J., etal., U.S. Pat. No. 5,604,123, issued 18 Feb. 1997; S. Zenno, et al, U.S.Pat. No. 5,618,722; K. V. Wood, U.S. Pat. No. 5,650,289, issued 22 Jul.1997; K. V. Wood, U.S. Pat. No. 5,641,641, issued 24 Jun. 1997; N.Kajiyama and E. Nakano, U.S. Pat. No. 5,229,285, issued 20 Jul. 1993; M.J. Cormier and W. W. Lorenz, U.S. Pat. No. 5,292,658, issued 8 Mar.1994; M. J. Cormier and W. W. Lorenz, U.S. Pat. No. 5,418,155, issued 23May 1995; de Wet, J. R., et al, Molec. Cell Biol. 7:725-737, 1987;Tatsumi, H. N., et al, Biochim. Biophys. Acta 1131:161-165, 1992; andWood, K. V., et al, Science 244:700-702, 1989; all herein incorporatedby reference. Another group of bioluminescent proteins includeslight-generating proteins of the aequorin family (Prasher, D. C., etal., Biochem. 26:1326-1332 (1987)). Luciferases, as well asaequorin-like molecules, require a source of energy, such as ATP,NAD(P)H, and the like, and a substrate, such as luciferin orcoelentrizine and oxygen.

Wild-type firefly luciferases typically have emission maxima at about550 nm. Numerous variants with distinct emission maxima have also beenstudied. For example, Kajiyama and Nakano (Protein Eng. 4(6):691-693,1991; U.S. Pat. No. 5,330,906, issued 19 Jul. 1994, herein incorporatedby reference) teach five variant firefly luciferases generated by singleamino acid changes to the Luciola cruciata luciferase coding sequence.The variants have emission peaks of 558 nm, 595 nm, 607 nm, 609 nm and612 nm. A yellow-green luciferase with an emission peak of about 540 nmis commercially available from Promega, Madison, Wis. under the namepGL3. A red luciferase with an emission peak of about 610 nm isdescribed, for example, in Contag et al. (1998) Nat. Med. 4:245-247 andKajiyama et al. (1991) Port. Eng. 4:691-693. The coding sequence of aluciferase derived from Renilla muelleri has also been described (mRNA,GENBANK Accession No. AY015988, protein Accession AAG54094).

In another aspect of the present invention, the light-generating proteinis a fluorescent protein, for example, blue, cyan, green, yellow, andred fluorescent proteins.

Several light-generating protein coding sequences are commerciallyavailable, including, but not limited to, the following. Clontech (PaloAlto, Calif.) provides coding sequences for luciferase and a variety offluorescent proteins, including, blue, cyan, green, yellow, and redfluorescent proteins. Enhanced green fluorescent protein (EGFP) variantsare well expressed in mammalian systems and tend to exhibit brighterfluorescence than wild-type GFP. Enhanced fluorescent proteins includeenhanced green fluorescent protein (EGFP), enhanced cyan fluorescentprotein (ECFP), and enhanced yellow fluorescent protein (EYFP). Further,Clontech provides destabilized enhanced fluorescent proteins (dEFP)variants that feature rapid turn over rates. The shorter half life ofthe dEFP variants makes them useful in kinetic studies and asquantitative reporters. DsRed coding sequences are available fromClontech DsRed is a red fluorescent protein useful in expressionstudies. Further, Fradkov, A. F., et. al., described a novel fluorescentprotein from Discosoma coral and its mutants which possesses a uniquefar-red fluorescence (FEBS Lett. 479 (3), 127-130 (2000)) (mRNAsequence, GENBANK Accession No. AF272711, protein sequence, GENBANKAccession No. AAG16224). Promega (Madison, Wis.) also provides codingsequences for fire fly luciferase (for example, as contained in the pGL3vectors). Further, coding sequences for a number of fluorescent proteinsare available from GENBANK, for example, accession numbers AY015995,AF322221, AF080431, AF292560, AF292559, AF292558, AF292557, AF139645,U47298, U47297, AY015988, AY015994, and AF292556.

Modified lux coding sequences have also been described, e.g., WO01/18195, published 15 Mar. 2001, Xenogen Corporation. In addition,further light generating systems may be employed, for example, whenevaluating expression in cells. Such systems include, but are notlimited to, Luminescent beta-galactosidase Genetic Reporter System(Clontech).

Positive selection markers include any gene which a product that can bereadily assayed. Examples include, but are not limited to, an HPRT gene(Littlefield, J. W., Science 145:709-710 (1964), herein incorporated byreference), a xanthine-guanine phosphoribosyltransferase (GPT) gene, oran adenosine phosphoribosyltransferase (APRT) gene (Sambrook et al.,supra), a thymidine kinase gene (i.e. “TK”) and especially the TK geneof the herpes simplex virus (Giphart-Gassler, M. et al., Mutat. Res.214:223-232 (1989) herein incorporated by reference), a nptII gene(Thomas, K. R. et al., Cell 51:503-512 (1987); Mansour, S. L. et al.,Nature 336:348-352 (1988), both references herein incorporated byreference), or other genes which confer resistance to amino acid ornucleoside analogues, or antibiotics, etc., for example, gene sequenceswhich encode enzymes such as dihydrofolate reductase (DHFR) enzyme,adenosine deaminase (ADA), asparagine synthetase (AS), hygromycin Bphosphotransferase, or a CAD enzyme (carbamyl phosphate synthetase,aspartate transcarbamylase, and dihydroorotase). Addition of theappropriate substrate of the positive selection marker can be used todetermine if the product of the positive selection marker is expressed,for example cells which do not express the positive selection markernptII, are killed when exposed to the substrate G418 (Gibco BRL LifeTechnology, Gaithersburg, Md.).

The vector typically contains insertion sites for insertingpolynucleotide sequences of interest, e.g., the novel EPX sequences ofthe present invention. These insertion sites are preferably includedsuch that there are two sites, one site on either side of the sequencesencoding the positive selection marker, luciferase and the promoter.Insertion sites are, for example, restriction endonuclease recognitionsites, and can, for example, represent unique restriction sites. In thisway, the vector can be digested with the appropriate enzymes and thesequences of interest ligated into the vector.

Optionally, the vector construct can contain a polynucleotide encoding anegative selection marker. Suitable negative selection markers include,but are not limited to, HSV-tk (see, e.g., Majzoub et al. (1996) NewEngl. J. Med. 334:904-907 and U.S. Pat. No. 5,464,764), as well as genesencoding various toxins including the diphtheria toxin, the tetanustoxin, the cholera toxin and the pertussis toxin. A further negativeselection marker gene is the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene for negative selection in 6-thioguanine.

The vectors described herein can be constructed utilizing methodologiesknown in the art of molecular biology (see, for example, Ausubel orManiatis) in view of the teachings of the specification. As describedabove, the vector constructs containing the expression cassettes areassembled by inserting the desired components into a suitable vectorbackbone, for example: a vector comprising (1) polynucleotides encodinga reporter protein, such as a light-generating protein, e.g., aluciferase gene, operably linked to a transcription control element(s)of interest derived from the mouse eosinophil peroxidase (EPX) genelocus; (2) a sequence encoding a positive selection marker; and,optionally (3) a sequence encoding a negative selection marker. Inaddition, the vector construct contains insertion sites such thatadditional sequences of interest can be readily inserted to flank thesequence encoding positive selection marker and luciferase-encodingsequence.

A preferred method of obtaining polynucleotides, suitable regulatorysequences (e.g., promoters) is PCR. General procedures for PCR as taughtin MacPherson et al., PCR: A PRACTICAL APPROACH, (IRL Press at OxfordUniversity Press, (1991)). PCR conditions for each application reactionmay be empirically determined. A number of parameters influence thesuccess of a reaction. Among these parameters are annealing temperatureand time, extension time, Mg2+ and ATP concentration, pH, and therelative concentration of primers, templates and deoxyribonucleotides.Exemplary primers are described below in the Examples. Afteramplification, the resulting fragments can be detected by agarose gelelectrophoresis followed by visualization with ethidium bromide stainingand ultraviolet illumination.

In one embodiment, PCR can be used to amplify fragments from genomiclibraries. Many genomic libraries are commercially available.Alternatively, libraries can be produced by any method known in the art.Preferably, the organism(s) from which the DNA is has no discernibledisease or phenotypic effects. This isolated DNA may be obtained fromany cell source or body fluid (e.g., eosinophil cells, ES cells, liver,kidney, blood cells, buccal cells, cerviovaginal cells, epithelial cellsfrom urine, fetal cells, or any cells present in tissue obtained bybiopsy, urine, blood, cerebrospinal fluid (CSF), and tissue exudates atthe site of infection or inflammation). DNA is extracted from the cellsor body fluid using known methods of cell lysis and DNA purification.The purified DNA is then introduced into a suitable expression system,for example a lambda phage. Another method for obtainingpolynucleotides, for example, short, random nucleotide sequences, is byenzymatic digestion.

Polynucleotides are inserted into vector backbones using methods knownin the art. For example, insert and vector DNA can be contacted, undersuitable conditions, with a restriction enzyme to create complementaryor blunt ends on each molecule that can pair with each other and bejoined with a ligase. Alternatively, synthetic nucleic acid linkers canbe ligated to the termini of a polynucleotide. These synthetic linkerscan contain nucleic acid sequences that correspond to a particularrestriction site in the vector DNA. Other means are known and, in viewof the teachings herein, can be used.

The vector backbone may comprise components functional in more than oneselected organism in order to provide a shuttle vector, for example, abacterial origin of replication and a eucaryotic promoter. Alternately,the vector backbone may comprise an integrating vector, i.e., a vectorthat is used for random or site-directed integration into a targetgenome.

The final constructs can be used immediately (e.g., for introductioninto ES cells or for liver-push assays), or stored frozen (e.g., at −20°C.) until use. In some embodiments, the constructs are linearized priorto use, for example by digestion with suitable restrictionendonucleases.

2.4 Transgenic Animals

The expression cassettes of the present invention may be introduced intothe genome of an animal in order to produce transgenic, non-humananimals for purposes of practicing the methods of the present invention.In a preferred embodiment of the present invention, the transgenicnon-human, animal may be a rodent (e.g., rodents, including, but notlimited to, mice, rats, hamsters, gerbils, and guinea pigs). When alight-generating protein is used as a reporter, imaging is typicallycarried out using an intact, living, non-human transgenic animal, forexample, a living, transgenic rodent (e.g., a mouse or rat). A varietyof transformation techniques are well known in the art. Those methodsinclude the following.

(i) Direct Microinjection into Nuclei: Expression cassettes can bemicroinjected directly into animal cell nuclei using micropipettes tomechanically transfer the recombinant DNA. This method has the advantageof not exposing the DNA to cellular compartments other than the nucleusand of yielding stable recombinants at high frequency. See, Capecchi,M., Cell 22:479-488 (1980).

For example, the expression cassettes of the present invention may bemicroinjected into the early male pronucleus of a zygote as early aspossible after the formation of the male pronucleus membrane, and priorto its being processed by the zygote female pronucleus. Thus,microinjection according to this method should be undertaken when themale and female pronuclei are well separated and both are located closeto the cell membrane. See, e.g., U.S. Pat. No. 4,873,191 to Wagner, etal. (issued Oct. 10, 1989); and Richa, J., (2001) “Production ofTransgenic Mice,” Molecular Biotechnology, March 2001 vol. 17:261-8.

(ii) ES Cell Transfection: The DNA containing the expression cassettesof the present invention can also be introduced into embryonic stem(“ES”) cells. ES cell clones which undergo homologous recombination witha targeting vector are identified, and ES cell-mouse chimeras are thenproduced. Homozygous animals are produced by mating of hemizygouschimera animals. Procedures are described in, e.g., Koller, B. H. andSmithies, O., (1992) “Altering genes in animals by gene targeting”,Annual review of immunology 10:705-30.

(iii) Electroporation: The DNA containing the expression cassettes ofthe present invention can also be introduced into the animal cells byelectroporation. In this technique, animal cells are electroporated inthe presence of DNA containing the expression cassette. Electricalimpulses of high field strength reversibly permeabilize biomembranesallowing the introduction of the DNA. The pores created duringelectroporation permit the uptake of macromolecules such as DNA.Procedures are described in, e.g., Potter, H., et al., Proc. Nat'l.Acad. Sci. U.S.A. 81:7161-7165 (1984); and Sambrook, ch. 16.

(iv) Calcium Phosphate Precipitation: The expression cassettes may alsobe transferred into cells by other methods of direct uptake, forexample, using calcium phosphate. See, e.g., Graham, F., and A. Van derEb, Virology 52:456-467 (1973); and Sambrook, ch.16.

(v) Liposomes: Encapsulation of DNA within artificial membrane vesicles(liposomes) followed by fusion of the liposomes with the target cellmembrane can also be used to introduce DNA into animal cells. SeeMannino, R. and S. Gould-Fogerite, BioTechniques, 6:682 (1988).

(vi) Viral Capsids: Viruses and empty viral capsids can also be used toincorporate DNA and transfer the DNA to animal cells. For example, DNAcan be incorporated into empty polyoma viral capsids and then deliveredto polyoma-susceptible cells. See, e.g., Slilaty, S. and H. Aposhian,Science 220:725 (1983).

(vii) Transfection using Polybrene or DEAE-Dextran: These techniques aredescribed in Sambrook, ch.16.

(viii) Protoplast Fusion: Protoplast fusion typically involves thefusion of bacterial protoplasts carrying high numbers of a plasmid ofinterest with cultured animal cells, usually mediated by treatment withpolyethylene glycol. Rassoulzadegan, M., et al., Nature, 295:257 (1982).

(ix) Ballistic Penetration: Another method of introduction of nucleicacid segments is high velocity ballistic penetration by small particleswith the nucleic acid either within the matrix of small beads orparticles, or on the surface, Klein, et al., Nature, 327, 70-73, 1987.

Any technique that can be used to introduce DNA into the animal cells ofchoice can be employed (e.g., “Transgenic Animal Technology: ALaboratory Handbook,” by Carl A. Pinkert, (Editor) First Edition,Academic Press; ISBN: 0125571658; “Manipulating the Mouse Embryo: ALaboratory Manual,” Brigid Hogan, et al., ISBN: 0879693843, Publisher:Cold Spring Harbor Laboratory Press, Pub. Date: September 1999, SecondEdition.). Electroporation has the advantage of ease and has been foundto be broadly applicable, but a substantial fraction of the targetedcells may be killed during electroporation. Therefore, for sensitivecells or cells which are only obtainable in small numbers,microinjection directly into nuclei may be preferable. Also, where ahigh efficiency of DNA incorporation is especially important, such astransformation without the use of a selectable marker (as discussedabove), direct microinjection into nuclei is an advantageous methodbecause typically 5-25% of targeted cells will have stably incorporatedthe microinjected DNA. Retroviral vectors are also highly efficient butin some cases they are subject to other shortcomings, as described byEllis, J., and A. Bernstein, Molec. Cell. Biol. 9:1621-1627 (1989).Where lower efficiency techniques are used, such as electroporation,calcium phosphate precipitation or liposome fusion, it is preferable tohave a selectable marker in the expression cassette so that stabletransformants can be readily selected, as discussed above.

In some situations, introduction of the heterologous DNA will itselfresult in a selectable phenotype, in which case the targeted cells canbe screened directly for homologous recombination. For example,disrupting the gene hart results in resistance to 6-thioguanine. In manycases, however, the transformation will not result in such an easilyselectable phenotype and, if a low efficiency transformation techniquesuch as calcium phosphate precipitation is being used, it is preferableto include in the expression cassette a selectable marker such that thestable integration of the expression cassette in the genome will lead toa selectable phenotype. For example, if the introduced DNA contains aneo gene, then selection for integrants can be achieved by selectingcells able to grow on G418.

Transgenic animals prepared as above are useful for practicing themethods of the present invention. Operably linking a promoter ofinterest to a reporter sequence enables persons of skill in the art tomonitor a wide variety of biological processes involving expression ofthe gene from which the promoter is derived. The transgenic animals ofthe present invention that comprise the expression cassettes of thepresent invention provide a means for skilled artisans to observe thoseprocesses as they occur in vivo, as well as to elucidate the mechanismsunderlying those processes.

With respect to transgenic animals carrying expression cassettes thatemploy a light-generating protein as a reporter sequence, the monitoringof expression of luciferase reporter expression cassettes usingnon-invasive whole animal imaging has been described (Contag, C. et al,U.S. Pat. Nos. 5,650,135, and 6,217,847, issued 22 Jul. 1997, and Apr.17, 2001, respectively, herein incorporated by reference in theirentireties; Contag, P., et al, Nature Medicine 4(2):245-247, 1998;Contag, C., et al, OSA TOPS on Biomedical Optical Spectroscopy andDiagnostics 3:220-224, 1996; Contag, C. H., et al, Photochemistry andPhotobiology 66(4):523-531, 1997; Contag, C. H., et al, MolecularMicrobiology 18(4):593-603, 1995). Such imaging typically uses at leastone photo detector device element, for example, a charge-coupled device(CCD) camera.

Thus, in one exemplary embodiment, transgenic mice carrying expressioncassettes comprising control elements derived from the mouse eosinophilperoxidase (EPX) gene locus operably linked to a luciferase-encodingreporter sequence may be used to monitor EPX promoter-mediatedexpression. The transgenic animals of the present invention thatcomprise the expression cassettes of the present invention also providea means for screening analytes that may be capable of modulatingexpression mediated by the mouse eosinophil peroxidase (EPX) genetranscriptional control elements.

Methods of administration of the analyte include, but are not limitedto, injection (subcutaneously, epidermally, intradermally), intramucosal(such as nasal, rectal and vaginal), intraperitoneal, intravenous, oralor intramuscular. Other modes of administration include oral andpulmonary administration, suppositories, and transdermal applications.Dosage treatment may be a single dose schedule or a multiple doseschedule. For example, the analyte of interest can be administered overa range of concentration to determine a dose/response curve. The analytemay be administered to a series of test animals or to a single testanimal (given that response to the analyte can be cleared from thetransgenic animal).

Thus, in one exemplary embodiment, transgenic mice carrying expressioncassettes comprising eosinophil peroxidase transcription controlelements, e.g., promoter, operably linked to a luciferase-encodingreporter sequence may be used to monitor the effects of a candidatecompound on eosinophil peroxidase transcription control elementmeditated-expression. Transgenic mice of the present invention may beused to screen compounds which may be effective pharmaceutical agents.

The creation and phenotypic characterization of transgenic animalscomprising eosinophil peroxidase gene derived transcription controlelements (i.e., a transgene) is described in Examples 2 and 3.

Criteria for selecting a transgenic animal, e.g., rodent, useful in amodel for screening compounds affecting the expression of, for example,the eosinophil peroxidase gene are generally as follows:

Criterion 1. Southern blot analysis and PCR analysis to identifytransgenic animals carrying the transgene (e.g., EPX transcriptioncontrol elements operably linked to coding sequences of interest, e.g.,sequences encoding a reporter gene, for example, a light generatingprotein).

Criterion 2. Eosinophils from the transgenic animal express the codingsequence of interest, e.g., a reporter gene, at a greater level thanother, non-eosinophil blood cell-types. Eosinophils as well as otherblood cell-types are isolated from transgenic animals. The cell-typesare fractionated (e.g., by FACs or panning) and reporter gene expressionin the different cell-types is evaluated.

Criterion 3 (may be optionally applied). Induction of expression of thecoding sequence of interest, e.g., a reporter gene, is observed byovalbumin challenge via intraperitoneal injection or via airwayinhalation. Induction is relative to basal levels of reporter geneexpression in the unchallenged transgenic animal.

Criterion 4 (may optionally be applied). Administration of IL-5 to thetransgenic rodent promotes trafficking of eosinophils, expressing thecoding sequence of interest, to the esophagus of said transgenic rodentrelative to other regions of the body of the living, transgenic rodent.When the reporter gene encodes a light-generating protein, suchtrafficking can be monitored by methods described in, e.g., Example 3.

Criterion 5 (may be optionally applied). Allergen-induced eosinophilcell recruitment to an air-pouch, e.g., a dorsal air pouch, of thetransgenic rodent is reduced by glucocorticoid treatment (e.g., levelsof expression of the reporter gene after allergen-induction are higherbefore treatment with dexamethasone than after treatment withdexamethasone). When the coding sequence of interest is a reporter genethat encodes a light-generating protein, such trafficking can bemonitored by methods described in, e.g., Example 3.

Criterion 6 (may be optionally applied). At baseline (i.e., a transgenicanimal maintained under healthy conditions), localization of expressionof the coding sequence of interest, e.g., a reporter gene, is to thelamina propria (e.g., of the stomach and intestines), that is, greaterexpression of reporter gene is seen in the lamina propria relative toother regions of the body of the transgenic animal. Alternately, or inaddition, greater expression of reporter gene in the lamina propriarelative to other regions of the body of the transgenic animal (e.g., inthe absence of interleukin (IL)-5 over-expression and/or oral allergenchallenge).

Criterion 7 (may be optionally applied). Induction of expression of thecoding sequence of interest, e.g., a reporter gene, is seen when IL-5 isover-expressed in the transgenic animal. IL-5 over-expression can beaccomplished in the transgenic animal, for example, via direct proteininjection, liver transfection (e.g., liver push experiments) orover-expression of IL-5 in the transgenic animal, where expression ofIL-5 may, for example, be mediated by a constitutive, inducible, orrepressible promoter.

The above-described optional phenotypic criteria may be applied, inaddition to criteria 1 and 2, to transgenic animal screening singly orin combinations. Typically, at least one of the optional phenotypiccriteria (e.g., criterion 3 and/or criterion 7) is applied for selectionof a suitable transgenic animal (which carries and expresses aEPX-reporter transgene).

In one exemplary embodiment, transgenic mice carrying expressioncassettes comprising the mouse eosinophil peroxidase (EPX) gene promoteroperably linked to a luciferase-encoding reporter sequence, and meetingat least criteria may be used to monitor the effects of a candidatecompound on EPX gene expression. The results of those experimentsdemonstrate that the transgenic mice of the present invention may beused to screen compounds which may be effective pharmaceutical agents.

2.5 Eosinophil Migration

Eosinophils are motile phagocytic cells that can migrate from blood intotissue spaces. It is believed that eosinophils play a role in defenseagainst parasitic organisms. For example, the secreted contents ofeosinophilic granules may damage membranes of parasites. However,although the release of these eosinophil-derived mediators may play aprotective role in parasitic infections, in response to allergens, thesemediators contribute to extensive tissue damage in late-phase reactions.The influx of eosinophils in the late-phase response has been shown tocontribute to the chronic inflammation of the bronchial mucosa thatcharacterizes persistent asthma. Eosinophils are known to be involved inseveral disease processes including, but not limited to, asthma, foodallergies, and atopic dermatitis.

Asthma is a multifactorial syndrome characterized by breathlessness,pulmonary constriction, mucous accumulation, and airwayhyper-reactivity. It is often, but not always associated with allergies(extrinsic) or environmental stimuli, e.g., tobacco smoke, but may alsobe induced by, for example, exercise or cold (intrinsic). Manypathophysiological manifestations of asthma are associated with airwayinfiltration by eosinophils and lymphocytes. Such infiltration ismediated by cytokines and chemokines. The extent of infiltrationgenerally correlates with the severity of disease. Leukocyte influx hasbeen associated with the development of lung dysfunction, even innominal cases of asthma. Antigen-induced mouse models of pulmonaryallergic disease have proved particularly informative in the geneticdissection of inflammatory pathways in the lung. Typically, these modelsinvolve sensitization with a specific antigen (e.g., ovalbumin) followedby airborne administration of the same antigen. Sensitized mice treatedwith aerosolized allergen develop leukocytic infiltrates of the airwaylumen dominated by CD4+ lymphocytes and eosinophils. These mice alsodevelop many of the changes indicative of asthma-related pathology,including airway hyper-responsiveness (AHR) and goblet cell hyperplasiatypically with accompanying excessive mucus production. Accordingly,employing eosinophils marked as described herein, migration ofeosinophils and other eosinophil responses may be studied in vivo inexperimental animals. Further, the transgenic animals of the presentinvention, particularly transgenic rodents carrying coding sequences fora light-generating protein under the control of EPX regulatorysequences, will facilitate the analysis of important components ofpro-inflammatory cascades that ultimately result in eosinophil airwayinfiltration and pathophysiological changes characteristic of asthma. Inaddition, the effects of selected analytes on eosinophil migration andresponse can be evaluated.

Cellular signals leading to airway inflammation, eosinophilinfiltration, and airway hyper-responsiveness have not yet beencompletely elucidated. Lymphocytes, eosinophils, and mast cells havebeen implicated in airway hyper-responsiveness of antigen-challenged,mouse models of asthma. For example, SCID mice, lacking both T and Blymphocytes, develop neither airway eosinophilia nor bronchialhyper-reactivity (Corry, et al., J. Exp. Med. 183:109 (1996)). Further,depletion of CD4+ lymphocytes (e.g., by treatment with anti-CD4antibodies or MHC Class II gene knock-outs) eliminated eosinophil airwayinfiltration and airway hyper-responsiveness in antigen-challenged mice(Garett, et al., Am. J. Respir. Cell & Mol. Biol. 10: 587 (1994)).However, depletion of CD8+ T lymphocytes with anti-CD8 antibodies had noeffect on lung eosinophil infiltration but eliminated airwayhyper-responsiveness (Nakajima, et al., Am. J. Respir. Cell & Mol. Biol.10:587 (1994); Hammelmann, et al., J. Exp. Med. 183:1719 (1996)). Italso appears that mast cells were not specifically required for eithereosinophil airway infiltration or airway hyper-responsiveness in a mousemodel (Bruselle, et al., Am. J. Respir. Cell & Mol. Biol. 12:254(1995)).

Transgenic animals which express a reporter, e.g., light-generatingprotein, under the transcriptional control of EPX regulatory elementscan provide models for eosinophil-related disease states, as well asmodels for the efficacy of therapeutic agents which can be useful totreat those diseases. For example, transgenic mice expressinglight-generating protein coding sequences under the transcriptionalcontrol of EPX regulatory elements can be useful as a model for diseasescharacterized by the presence of pulmonary eosinophilic infiltrations,including, but not limited to, the following: asthma (extrinsic orintrinsic), pulmonary eosinophilia, Loffler's syndrome, eosinophilicpneumonia, eosinophilic myalgia, atopic disease, e.g., allergies orasthma, emphysema, pulmonary fibrosis, Wegener's granulomatosis,lymphoidmatoid granulomatosis, eosinophilic leukemia, eosinophilicgranuloma of the lung, adult respiratory distress syndrome, andpost-trauma pleural effusions which contain eosinophils or eosinophilcontaining pleural effusions associated with infections, such astuberculosis (see Spry, In: Eosinophils, Oxford University Press, pp.205-212 (1988)).

The transgenic animals of the present invention, e.g., transgenic miceexpressing light-generating protein coding sequences under thetranscriptional control of EPX regulatory elements, can be useful as amodel for atopic dermatitis, eosinophilic fasciitis, eosinophiliamyalgia, contact hypersensitivity diseases, or other skin allergic orhypersensitivity reaction.

Furthermore, such transgenic animals can be employed as vehicles to testagents which are known to, or which may be useful to, reduce, inhibit,or other wise affect eosinophil migration. Agents which inhibiteosinophil-associated pathologies are known to the art and their in vivoeffects can be evaluated using the transgenic animals of the presentinvention. In addition, new agents can be identified that affecteosinophil-associated pathologies employing the recombinant cells andtransgenic animals described herein.

2.6 Screening Analytes

The methods of monitoring promoter activity discussed above may beemployed for the purpose of screening analytes (e.g., candidate drugs)which modulate a variety of biological processes associated withexpression of the mouse eosinophil peroxidase (EPX) gene. Screening maybe accomplished by means of in vitro assays employing transiently orstably transfected cells, and may also be conducted using the transgenicanimals of the present invention discussed above, either by themselvesor in conjunction with other wild-type or transformed cells or tissuesthat have been introduced into those animals.

In one aspect of the invention, analytes which affect eosinophilmigration or otherwise affect eosinophil-response in eosinophil-relatedpathologies (see above) can be screened for their affects using therecombinant cells and/or transgenic animals of the present invention.For example, the effects of an analyte can be evaluated in transgenicmice expressing light-generating protein coding sequences under thetranscriptional control of EPX regulatory elements and eosinophilmigration to the site of inflammation can be monitored in vivo, in realtime, with and without treating the animal with the analyte. Suchevaluation of analytes allows the identification of analytes useful,e.g., for the treatment of asthma.

The particular assay method used to measure the effects of variouscandidate compounds on eosinophil migration or EPX promoter activitywill be determined by the particular reporter sequence present in theexpression cassette carried by the cells or animals employed. Asdiscussed above, promoter activity in transgenic animals carryingconstructs employing reporter sequences encoding light-generatingproteins may be measured by means of ex vivo assay methods or by meansof the in vivo bioluminescent imaging technique reference previously. Ananimal eosinophil migration model will greatly enhance the process ofvalidating analytes that are useful in the management ofeosinophil-related disease states, that is, disease states whereeosinophils serve as a marker.

In a further aspect of this invention, the expression cassettes andvectors comprising mouse eosinophil peroxidase (EPX) transcriptioncontrol element sequences can be used to facilitate eosinophilperoxidase (EPX) gene expression regulation studies in vitro and invivo. For example, regulatory sequences involved in the cell-typespecific expression of the EPX gene can be identified. Specificlocations of selected transcriptional control elements within a definedpolynucleotide sequence can be identified by methods known to those ofskill in the art, e.g., sequence comparison, deletion analysis, and/orlinker-insertion mutagenesis, in view of the teachings of the presentspecification. Identification of regulatory sequences associated withcell-type specific expression allows, for example, the use of suchregulatory sequences to confer cell-type specific expression to otherpromoters, e.g., a basal promoter.

For screening purposes, eosinophil cells may be transformed with anexpression vectors comprising a reporter gene (e.g., luciferase)operably linked to the EPX gene promoters of this invention. Thetransformed cells are next exposed to various test substances and thenanalyzed for expression of the reporter gene. The expression exhibitedby these cells can be compared to expression from cells that were notexposed to the test substance. A compound that modulates the promoteractivity of the EPX promoter will result in modulated reporter geneexpression relative to the control.

Thus, one aspect of the invention is to screen for test compounds thatregulate (i.e., stimulate or inhibit) gene expression levels mediated bythe EPX-locus derived transcription control elements (e.g., promoters).Screening may be accomplished by, for example, (i) contacting host cellsin which the EPX promoter disclosed herein is operably linked to areporter gene with a test medium containing the test compound underconditions which allow for expression of the reporter gene; (ii)measuring the expression of the reporter gene in the presence of thetest medium; (iii) contacting the host cells with a control medium whichdoes not contain the test compound but is otherwise essentiallyidentical to the test medium in (i), under conditions essentiallyidentical to those used in (i); (iv) measuring the expression ofreporter gene in the presence of the control medium; and (v) relatingthe difference in expression between (ii) and (iv) to the ability of thetest compound to affect the activity of the promoter.

In a further aspect, the present invention provides methods of measuringthe ability of a test compound to modulate EPX transcription by: (i)contacting a host cell in which the EPX promoter, disclosed herein, isoperably linked to a reporter gene with an inducer of the promoteractivity under conditions which allow for expression of the reportergene; (ii) measuring the expression of the reporter gene in the absenceof the test compound; (iii) exposing the host cells to the test compoundeither prior to, simultaneously with, or after contacting, the hostcells with the inducer; (iv) measuring the expression of the reportergene in the presence of the test compound; and (iv) relating thedifference in expression between (ii) and (iv) to the ability of thetest compound to modulate EPX-mediated transcription.

Various forms of the different embodiments of the invention, describedherein, may be combined.

Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Materials and Methods

Unless indicated otherwise, the experiments described herein wereperformed using standard methods.

A. PCR Amplification

For PCR amplifications, the reaction mix contained: 5 μl of 10× reactionbuffer (no MgCl₂); 4 μl 25 mM MgCl₂; 0.4 μl of 25 mM dNTP mix; 0.5 μl of10 pmoles/ul forward primer; 0.5 μl of 10 pmoles/μl reverse primer; 1 μl(0.2 μg) of DNA (BAC or genomic); 38.35 μl of H₂O; and 0.25 μl of TaqPolymerase (Life Technologies). The PCR was carried out as follows: 3minutes at 94° C.; 35 cycles of 94° C., 45 seconds, 60° C., 45 seconds,72° C. 45 seconds 7 minutes at 72° C.; and at 4° C.

B. Southern Blotting

(i) Primers were designed and used to PCR screen a mouse 129/SvJ genomicDNA BAC (bacterial artificial chromosome) library (Genome Systems, Inc.,St. Louis, Mo.) in order to isolate novel mouse eosinophil peroxidase(EPX) gene promoter sequences.

A library containing, on average, contained inserts of 120 kb with sizesranging between 50 kb to 240 kb was screened. A large genomic DNAfragment that contained EPX promoter region was obtained.

The EPX BAC DNA was isolated by CsCl ultracentrifugation and digestedwith various restriction enzymes for 2 hours. Digested DNA fragmentswere separated on a 1% agarose gel. The gel was depurinated in 250 mMHCL for 10 minutes and then denatured twice in 20×SSC with 0.5M NaOH for20 minutes. DNA was then transferred onto Hybond N+ membrane (Amersham,Piscataway N.J.) with 20×SSC for 1-2 hours using a vacuum blottingapparatus (Stratagene, La Jolla, Calif.). After transferring, themembrane was cross-linked according to the manufacturer's directionsusing UV Cross-Linker (Stratagene, La Jolla, Calif.) and rinsed with5×SSC. The membrane was then prehybridized at 60° C. for 1-6 hours withprehybridization solution (Stratagene, La Jolla, Calif.).

Probes were prepared by labeling PCR fragments or isolated DNA. Afterhybridization, the membrane was washed twice with pre-warmed 1×SSC, 0.1%SDS for 20 minutes at 60° C. each time. Subsequently, the membrane waswashed twice with pre-warmed 0.5×SSC for 20 minutes at 60° C. each time.The membrane was blocked at RT for 1 hour using blocking solution(Stratagene, La Jolla, Calif.) and incubated with antibody conjugated toalkaline phosphatase for 1 hour. After three washed, substrate CDP-Starwas added for 5 minutes. The membrane was exposed to X-ray film forbetween 1 minute and 3 hours.

C. Preparation of Transgenic Animals

The transgenic animals described below were prepared using themicroinjection into single cell stage embryos (see, e.g., U.S. Pat. No.4,873,191 to Wagner, et al. (issued Oct. 10, 1989); Richa, J., (2001)Molecular biotechnology 17:261-8. The embryos were implanted intopseudo-pregnant females and the offspring screened by PCR using primerslucF1 (GCCATTCTATCCGCTGGAAGATGG; SEQ ID NO:3) and lucR4(CGATTTTACCACATTTGTAGAGGTTTTACTTGC; SEQ ID NO:4). Imaging of animals wasdone as described herein.

D. In Vivo Imaging

In vivo imaging was performed as described previously (Contag, et al.(see e.g., Contag, P. R., et al., (1998) Nature Med. 4:245-7; Contag, C.H., et al., (1997) Photochem Photobiol. 66:523-31; Contag, C. H., etal., (1995) Mol Microbiol. 18:593-603); Zhang et al., (2001) TransgenicRes. 2001 Oct; 10(5):423-34) using either an intensified CCD camera(ICCD; model C2400-32, Hamamatsu, Japan) fitted with a 50 mm f 1.2Nikkor lens (Nikon, Japan) and an image processor (Argus 20, Hamamatsu),or with a cryogenically cooled camera (Roper Scientific, Trenton, N.J.)fitted with a 50 mm f 0.95 Navitar lens (Buhl Optical, Pittsburgh, Pa.)available as an integrated imaging system (IVIS™ (Xenogen Corporation,Alameda, Calif.) Imaging System) controlled using LivingImage® (XenogenCorporation, Alameda, Calif.) software.

The substrate luciferin was injected into the intraperitoneal cavity ata dose of 150 mg/kg body weight (30 mg/ml Luciferin stock) approximatelyfive minutes prior to imaging. Mice were anesthetized with eitherNembutal (25-50 mg/kg body weight) or in a gas chamber with anisoflurane/oxygen mixture and isoflurane tubing was placed on theanimals' noses, and placed on the imaging stage under anesthesia. Micewere imaged from the ventral side for 1 minute. Relative photon emissionover the liver region was quantified using LivingImage® software(Xenogen, Alameda, Calif.).

EXAMPLE 1 Isolation of Mouse Eosinophil Peroxidase TranscriptionalControl Element Sequences and Vector Construction

A pair of PCR primers specific for the mouse EPX promoter was designed(Primer 1, 5′-tgcatccatgaacccaagactaga-3′, SEQ ID NO:10; Primer 2,5′-cccactacagctaagcaggcaagca-3′, SEQ ID NO: 11). PCR conditions weretested for specificity using the PCR conditions described above.

The PCR reaction using these primers was used to screen the BAC librarydescribed above. Two BAC clones were identified. One of the clones(MEPOXBA11k) was confirmed to comprise the EPX transcription controlelements by sequencing. The plasmid MEPOXBA11k was sequenced from bothends and the sequence from one end matched the known sequence of themouse EPX cDNA (GENBANK Accession No. D78353).

An XbaI fragment (10,985 bp) from the BAC was cloned intopBluescript-SK. Physical mapping was done on this plasmid. A restrictionmap of this clone is presented in FIG. 2. The sequence of 9,828nucleotides of this clone, located upstream of the initiating ATG of theEPX gene, was determined. The sequence is presented in FIG. 1A (SEQ IDNO:1). A novel approximately 9.5 kb region of the EPX gene locus wasidentified from nucleotide position 1 to 9,450 of FIG. 1A and thatapproximately 9.5 kb sequence is presented alone in FIG. 1B (SEQ IDNO:2).

A vector for use in the generation of transgenic, non-human animals, wasconstructed as follows. An approximately 9.8 kb restriction fragmentfrom the XbaI 10,985 bp clone was isolated by digestion with XhoI andpartial digestion with BsrGI. The resulting approximately 9.8 kbfragment was cloned into the pGL3-Intron-Basic vector upstream of thehuman globin intron II which was upstream of firefly luciferase codingsequences (see below). The resulting vector was designated MEPO-luc. Thesequence of the approximately 9.8 kb fragment corresponds to nucleotidepositions 1-9,757 of FIG. 1A and are designated as SEQ ID NO:7.

The pGL3-Intron-Basic vector was constructed essentially as follows. An861 bp fragment (including 849 bp human globin intron II and 12 bpexon-intron boundary sequence) was amplified by PCR from human genomicDNA. The accuracy of the intron, amplified product was confirmed bysequencing. The primers used were as follows: PCR Primer 1:5′-agtcaagcttcagggtgagtctatgggacccttg-3′ (SEQ ID NO:5); PCR Primer 2:5′-gactaagcttaggagctgtgggaggaagataagag-3′ (SEQ ID NO:6). A yellow-greenluciferase with an emission peak of about 540 nm is commerciallyavailable in a plasmid vector from Promega, Madison, Wis. under the namepGL3basic. The PCR human globin intron II fragment was cloned intoHindIII site of pGL3basic and the resulting vector was designatedpGL3-Intron-Basic.

TABLE 1 Approximate Size Starting Position Ending of Fragment from ofEPX gene Position of EPX Vector the EPX gene locus fragment gene locusfragment Name locus relative to FIG. 1A relative to FIG. 1A MEPO-luc9757 bp 1 9757

This vector construct was used to generate transgenic mice as describedin Example 2.

EXAMPLE 2 Transgenic Animals

Transgenic mice were generated essentially as follows. The MEPO-lucplasmid was digested with NotI and XhoI. The resulting fragmentcontaining mouse EPX promoter and luciferase was separated bysize-fractionation using an agarose gel. The fragment was then isolatedfrom the agarose gel. The purified DNA was injected into fertilized eggsof FVB mice (Charles River Laboratories, Inc., Wilmington, Mass.). Thetransgenic lines were created by the microinjection method (see, e.g.,U.S. Pat. No. 4,873,191 to Wagner, et al. (issued Oct. 10, 1989); andRicha, J., (2001) “Production of transgenic mice” Molecularbiotechnology March 2001 vol. 17:261-8) using FVB donor embryos. Theinjected eggs were transplanted into pseudo-pregnant female foster micefollowing standard procedures (e.g., Methods in Enzymology, volume 225,page 747-771, edited by Paul M. Wassarman & Melvin L. DePamphilis.). Theresulting mice were screened for the presence of the EPX/luciferasesequences by PCR as described above. Alternately, the founder mice arescreened by PCR using luciferase primers LucF1 and LucR4 or primers Luc3 primer (5′-GAAATGTCCGTTCGGTTGGCAGAAGC-3′ (SEQ ID NO:8)) and Luc 4primer (5′-CCAAAACCGTGATGGAATGGAACAACA-3′ (SEQ ID NO:9)). These sameprimers may also be used to screen Tg offspring.

Transgenic founders and transgenic offspring may also be evaluated forthe presence and location of the transgene (e.g., an EPX transcriptioncontrol element operably linked to a reporter gene, for example, alight-generating protein) using Southern Hybridization Analysis.

For example, the 1.8 kb HindIII/XbaI fragment from pGL3-Basic containingthe entire luciferase cDNA (Promega Corp.) is used as probe for Southernhybridization. Ten μg of heterozygous genomic DNA is digested with aselected restriction enzyme (e.g., BamHI) and 17 pg of pGL3-Basic wasloaded as a positive control. The expected size of transgene iscalculated based on the known restriction map of the construct used togenerate the transgenic animals. Results from such Southern analysis canbe used to demonstrate the presence of the transgene in the transgenicmice.

These mice are then used as founders for a transgenic colony.

EXAMPLE 3 Phenotypic Data as Applied to Selection Criteria

General Methods

In vivo imaging was performed as described previously (Contag, et al.(see e.g., Contag, P. R., et al., (1998) Nature Med. 4:245-7; Contag, C.H., et al., (1997) Photochem Photobiol. 66:523-31; Contag, C. H., etal., (1995) Mol Microbiol. 18:593-603); Zhang et al., (2001) TransgenicRes. 2001 Oct; 10(5):423-34) using either an intensified CCD camera(ICCD; model C2400-32, Hamamatsu, Japan) fitted with a 50 mm f 1.2Nikkor lens (Nikon, Japan) and an image processor (Argus 20, Hamamatsu),or with a cryogenically cooled camera (Roper Scientific, Trenton, N.J.)fitted with a 50 mm f 0.95 Navitar lens (Buhl Optical, Pittsburgh, Pa.)available as an integrated imaging system (IVIS™ Imaging System,Xenogen, Corporation, Alameda, Calif.) controlled using LivingImage®software (Xenogen, Corporation, Alameda, Calif.).

The substrate luciferin was injected into the intraperitoneal cavity ata dose of 150 mg/kg body weight (30 mg/ml Luciferin stock) approximatelyfive minutes prior to imaging. Mice were anesthetized with eitherNembutal (25-50 mg/kg body weight) or in a gas chamber with anisoflurane/oxygen mixture and isoflurane tubing was placed on theanimals' noses, and placed on the imaging stage under anesthesia. Micewere typically imaged from the ventral side for 1 minute. Relativephoton emission was quantified using LivingImage® software (Xenogen,Alameda, Calif.).

These imaging methods can be used to track events in a test subject overtime. For example, a compound may be administered to a subject(comprising a light-generating reporter), and photon emission from thesubject before, during, and/or after administration of the compound maybe measured. Such measuring may be repeated at selected time intervalswhich is typically effective to track an effect of the compound on alevel of reporter expression in the subject over time.

General methods for evaluating the transgenic animal lines were asfollows. Tg founders were bred to wild-type FvB mice to generate F1mice. A female transgenic founder is typically bred to a wild-type FvBmale and a male transgenic founder is typically bred to a few wild-typeFvB females.

A Luciferin stock solution of 30 mg/ml was prepared in sterile PBS.Luciferin was purchased as D-Luciferin Potassium Salt, as Cat #XR-1001,from Biosynth AG, Switzerland.

Dexamethasone (Cat #D1756), may be purchased from Sigma (St. Louis, Mo.)and may be prepared in a solution of DMSO and injected IP at a dose ofbetween about 1-150 mg/kg. DMSO is administrated as a vehicle control.The duration of treatment with dexamethasone is typically for hours, 2-3days, up to about 7-10 days. Alternately, dexamethasone may besubcutaneously administered (see, e.g., Das, A. M., et al., Br JPharmacol. 1997 May, 121(1):97-104, herein incorporated by reference).

The route of administration for luciferin is, typically, IP. The dose ofreagent administration of luciferin substrate was as follows. Dose ofluciferin: 150 mg/kg of a 30 mg/ml luciferin stock was injected IP fiveminutes before imaging in the IVIS™ (Xenogen Corporation, Alameda,Calif.) system.

Following luciferin administration the animals were anesthetized usinggas anesthesia (Isoflurane) and placed in an IVIS™ box (XenogenCorporation, Alameda, Calif.) for imaging. All animals were imagedbefore and after chemical administration, and imaged at high resolution(binning 2).

Phenotyping

As discussed above, the following phenotypic criteria are applied to theselection of transgenic animals for use in methods of the presentinvention:

Criterion 1. Southern blot analysis and PCR analysis to identifytransgenic animals carrying the transgene (e.g., EPX transcriptioncontrol elements operably linked to sequences encoding luciferase).

Southern blot analysis and PCR analysis are performed essentially asdescribed above.

Criterion 2. Eosinophils from the transgenic animal express luciferaseat a greater level than other, non-eosinophil blood cell-types.Eosinophils as well as other blood cell-types are isolated fromtransgenic animals. The cell-types are fractionated (e.g., by FACs orpanning) and reporter gene expression in the different cell-types isevaluated.

Methods of cell fractionation (e.g., FACs and/or panning) are known inthe art (e.g., Shinagawa, K., and Anderson, G. P., J. Immunol. Methods200 Apr. 3, 237(1-2):65-72; Fattah, D., et al., Cytokine 1996 March8(3):248-259; Hoang, T., et al., Blood 1983 March 61(3):580-588; Hunt,T. C., et al., Clin. Exp. Allergy 1993 May 23(5):425-434; Burgess, A.W., et al., Exp. Hematol. 1980 Jan 8(1): 108-109; all hereinincorporated by reference in their entireties) and may be applied tophenotyping in view of the teachings of the present specification.

Criterion 3 (may be optionally applied). Induction of luciferase signalis observed by ovalbumin challenge via intraperitoneal injection or viaairway inhalation. Ovalbumin challenge models are known in the art andmay be applied to phenotyping in view of the teachings of the presentspecification. See, for example, Liu C, Wang Z, Liang Z, Lei S, Chin MedJ (Engl) 2000 September 113(9):783-6; Tomkinson A, Duez C, Cieslewicz G,Pratt J C, Joetham A, Shanafelt M C, Gundel R, Gelfand E W, J ImmunolMay 1, 2001 166(9):5792-800; Tomkinson A, Cieslewicz G, Duez C, Larson KA, Lee J J, Gelfand E W, Am J Respir Crit Care Med 2001 March 163(3 Pt1):721-30; Cui X, Guo Z, Xu W, Chen Y, Zhu Y, Chin Med J (Engl) 1998October 111(10):940-4; Trifilieff A, El-Hashim A, Bertrand C, Am JPhysiol Lung Cell Mol Physiol 2000 December 279(6):L1120-8; TrifilieffA, El-Hasim A, Corteling R, Owen C E, Br J Pharmacol 2000 August130(7):1581-8; Blyth D I, Wharton T F, Pedrick M S, Savage T J, SanjarS, Am J Respir Cell Mol Biol 2000 August 23(2):241-6; Dohi M, TsukamotoS, Nagahori T, Shinagawa K, Saitoh K, Tanaka Y, Kobayashi S, Tanaka R,To Y, Yamamoto K, Lab Invest 1999 December 79(12):1559-71; all hereinincorporated by reference in their entireties. Induction is relative tobasal levels (i.e., pre-ovalbumin challenge) of luciferase expression inthe unchallenged transgenic animal.

Criterion 4 (may optionally be applied). Administration of IL-5 to thetransgenic rodent promotes greater trafficking of eosinophils to theesophagus of said transgenic rodent than to other regions of the body ofthe living, transgenic rodent (see, e.g., Mishra, A., J. Immunol. 2002Mar. 1, 168(5):2462-2469; Mishra, A., et al., J. Clin. Invest. 2001,107:83-90, both herein incorporated by reference). The trafficking ismonitored by tracking expression of the coding sequence of interest(e.g., luciferase). Such tracking can be performed over time. Luciferaseexpression in intact, living, transgenic rodents can be monitored asdescribed herein above.

Criterion 5 (may be optionally applied). Allergen-induced eosinophilcell recruitment to an air-pouch (e.g., dorsal air pouch) of thetransgenic rodent is reduced by glucocorticoid treatment (see, forexample, Das, A. M., et al., Br J Pharmacol. 1997 May, 121(1):97-104,herein incorporated by reference). In one embodiment, levels ofexpression of luciferase after allergen-induction are higher beforetreatment with dexamethasone than after treatment with dexamethasone.Dexamethasone may be administered, for example, at an amount of betweenabout 1-150 mg/kg body weight of the transgenic animal, preferablybetween about 2-10 mg/kg body weight. The dexamethasone can beadministered following a predetermined treatment schedule. Dexamethasonecan be administered IP or subcutaneously. Use of luciferase as areporter gene permits such trafficking to be monitored by methodsdescribed herein above. The reduced recruitment to the air-pouch isobserved, for example, by a reduction of the level of expression of thecoding sequence of interest (e.g., luciferase) in the region of the bodyof the transgenic animal corresponding to the air-pouch.

Criterion 6 (may be optionally applied). At baseline (i.e., a transgenicanimal maintained under healthy conditions), expression of luciferase islocalized to the lamina propria (typically of the stomach andintestines), that is, greater basal expression of luciferase is seen inthe lamina propria relative to basal expression in other regions of thebody of the transgenic animal. Alternately, or in addition, greaterexpression of luciferase may be seen in the lamina propria relative toother regions of the body of the transgenic animal, for example, in theabsence of interleukin (IL)-5 over-expression and/or oral allergenchallenge. In addition, levels of eosinophils can be induced by antigenexposure under Th2 conditions (see, for example, Rothenberg, M. E., etal., Immunol. Rev. 2001 Febuary, 179:139-155, herein incorporated byreference).

Criterion 7 (may be optionally applied). Induction of luciferaseexpression in the transgenic rodent is seen when IL-5 is over-expressedin the transgenic animal (relative to luciferase expression when IL-5 isnot over-expressed). Van Oosterhout, A J, et al. (Am Rev Respir Dis 1993Mar, 147(3):548-52, herein incorporated by reference) describe thattreatment with IL-5 for seven days tends to increase the number ofeosinophils in bronchoalveolar lavage (BAL) fluid. IL-5 over-expressioncan be accomplished in the transgenic animal, for example, via directprotein injection, liver transfection (e.g., liver push experiments) orover-expression of IL-5 in the transgenic animal, where expression ofIL-5 may, for example, be mediated by a constitutive, inducible, orrepressible promoter.

Coding sequences of IL-5 are known for many animals (e.g., Campbell, H.D., et al., Eur. J. Biochem. 174 (2), 345-352 (1988); and GenBankAccession Nos. AJ01299, U34588, and X06271). Suitable expressionplasmids/vectors are known in the art. For liver push experiments,plasmids may be administered by intravenous injection according to themethod of Liu F., et al., (1999) Human Gene Therapy 10: 1735-1737. Forexample, a 2.2 ml of a PBS solution containing the desired IL-5containing-constructs are injected into the tail vein over a period ofless than 8 seconds.

The above-described optional phenotypic criteria may be applied, inaddition to criteria 1 and 2, to transgenic animal phenotypic screeningsingly or in combinations. Typically, at least one of the optionalphenotypic criteria (e.g., criterion 3 and/or criterion 7) is appliedfor selection of a suitable transgenic animal (which carries andexpresses a EPX-luciferase transgene).

Accordingly, transgenic rodents (e.g., mice), comprising EPXtranscription control elements operably linked to luc coding sequences,having desirable phenotypes, as outlined in the above criteria, can beidentified by the methods taught herein.

The eosinophils of the resulting transgenic rodents are labeled withluciferase (i.e., expression of luciferase is mediated byeosinophil-specific eosinophil peroxidase transcription controlelements). This transgenic rodent model is used to monitor eosinophilsin vivo, for example, employing methods of inducing disease conditionslike Asthma, Chronic eosinophilic pneumonia, Allergic rhinitis andAtopic dermatitis. The inducing agents employed can be either biologicalmolecules or chemical compounds. These transgenic rodents are then usedas models to, for example, evaluate the effect of test compounds on suchdisease states.

As is apparent to one of skill in the art, various modification andvariations of the above embodiments can be made without departing fromthe spirit and scope of this invention. These modifications andvariations are within the scope of this invention.

1. A transgenic mouse whose genome comprises an expression cassettecomprising a polynucleotide having nucleotides 1 to 9,825 of SEQ ID NO:1or variants thereof that have at least 95% or greater identity tonucleotides 1-9,825 of SEQ ID NO:1 over then entire length ofnucleotides 1-9,825, operably linked to a sequence encoding alight-generating protein, wherein the polynucleotide causes increasedexpression of the light-generating protein in eosinophils of thetransgenic mouse as compared to non-eosinophil blood cell-types.
 2. Thetransgenic mouse of claim 1, wherein said polynucleotide has at least95% or greater identity to SEQ ID NO:2.
 3. The transgenic mouse of claim1, wherein said polynucleotide has at least 95% or greater identity toSEQ ID NO:7.
 4. The transgenic mouse of claim 1, wherein saidpolynucleotide consists of a polynucleotide having at least 95% orgreater identity to nucleotides 1-9,825 of SEQ ID NO:1.
 5. Thetransgenic mouse of claim 2, wherein said polynucleotide consists of apolynucleotide having at least 95% or greater identity to SEQ ID NO:2.6. The transgenic mouse of claim 3, wherein said polynucleotide consistsof a polynucleotide having at least 95% or greater identity to SEQ IDNO:7.
 7. The transgenic mouse of claim 1, wherein ovalbumin challengevia intraperitoneal injection or via airway inhalation inducesexpression of the light generating protein.
 8. The transgenic mouse ofclaim 1, wherein administration of IL-5 to the transgenic mouse promotesgreater trafficking of eosinophils to the esophagus of said transgenicmouse than to other regions of the body of said transgenic mouse andsaid trafficking is monitored by tracking expression of the lightgenerating protein.
 9. The transgenic mouse of claim 1, wherein greaterbasal expression of the light generating protein is detected at greaterlevels in the lamina propria relative to basal expression of the lightgenerating protein in other regions of the body of the transgenic mouse.10. The transgenic mouse of claim 1, wherein expression of the lightgenerating protein is induced in the transgenic mouse when IL-5 isover-expressed in the transgenic mouse.
 11. The transgenic mo-use ofclaim 1, wherein levels of expression of the light generating proteinafter allergen-induction are higher before treatment with aglucocorticoid than after treatment with the glucocorticoid.
 12. Thetransgenic mouse of claim 11, wherein said glucocorticoid isdexamethasone.
 13. The transgenic mouse of claim 1, wherein thelight-generating protein is a bioluminescent protein or a fluorescentprotein.
 14. The transgenic mouse of claim 13, wherein thebioluminescent protein is luciferase.
 15. The transgenic mouse of claim13, wherein the fluorescent protein is selected from the groupconsisting of blue fluorescent protein, cyan fluorescent protein, greenfluorescent protein, yellow fluorescent protein, and red fluorescentprotein.